© 2014 Nature America, Inc. All rights reserved. viability. The involvement of a target of interest in the functional in the functional interest of a of target The involvement viability. cellular on impact or targets downstream of status phorylation readout,functional such a as altered on levels of metabolites, changes in based the phos is compound a to response the which in assays phenotypic to contrast in much target, a to ligand of ing bind the of measure direct a is engagement target addition, In CETSA methods complement similar studies on purified proteins. thereby the biological relevance of the studied system. As such, the and and biomolecules, proteins other with and tions interactions modifica post-translational localization, subcellular correct the understanding, our of best the to preserve, helps cells primary tissues more in complex environments such as level in cell lysates, cells intact and even target the at investigated is stabilization induced tional conven TSAs, it although greatly broadens as the utility, as the ligand- principle same the on builds approach CETSA The proteins of groups of members among specificities ligand deconvolute effectively to means a as initiatives, genomics tural in, struc TSAs e.g., of use extensive made has research academic techniques scattering–based light or fluorescence- e.g., using, targets isolated on ligands stabilizing of presence for the screened be to libraries compound allowing decade, a than more for industry discovery drug the in value great of been have equilibrium at systems isolated for properties, thermodynamic energies, other as free well as binding of estimation the for used be can which binding, ligand of a result as protein to the inferred stabilization interest a within cell of target to a protein binds directly and reaches candidate a drug We developed CETSA to monitor and quantify the extent to which I Both approaches can be completed in a day. protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. O proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates exemplified with experimental data on the human kinases p38 thermal shift assay ( a proof-of-principle study describing the ofimplementation thermal shift assays in a cellular format, which we call the cellular extensively on purified proteins in the drug discovery industry and in academia to detect interactions. T Published online 7 August 2014; Institutet, Solna, Sweden. Correspondence should be addressed to T.L. ( Science for Stockholm, Life Laboratory Translationalof Division Medicine and Chemical Biology, Medical and of Biophysics,Biochemistry Department Karolinska 1 Pär Nordlund Rozbeh Jafari target interactions in cells The cellular thermal shift assay for evaluating drug 2100 knowledge prior on based and selective, sufficiently are these if modulators, known of use the through inferred be can response Department of Medical and of Biophysics,Biochemistry Department Biophysics,of Division Karolinska Institutet, Stockholm, Sweden. NTRO hermal hermal shift assays are used to study thermal ofstabilization proteins upon ligand binding. protocol ne ne approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly
| VOL.9 NO.9VOL.9 1 D . The possibility to directly study target engagement in in engagement target study directly to possibility The . UCT I ON 2– 1 1 5 , Helena Almqvist | & MolinaDaniel Martinez 2014 . Melting temperature ( temperature .Melting 1 . It relies on the of thermodynamic principle CETSA | natureprotocols doi:10.1038/npr ). ). T he he method allows studies of target engagement of drug candidates in a cellular context, herein 2 , Hanna Axelsson ot.2014.13 T m ) shift assays (TSAs) (TSAs) assays shift ) 8 1 6– 9 . Similarly, Similarly, . [email protected] 2 , Marina Ignatushchenko 10,1 1 a - - - - - . and lows denaturation of the target protein of choice of protein target the of denaturation lows fol that aggregation irreversible the on rely such, procedures As CETSA samples. control and ligand-treated for temperatures different at protein target soluble remaining of amount the ing resistance. treatment monitor even and dosing adjust schemes, developed as a means to select treatment for patients, set treatment in tool clinic the diagnostic a as and trials clinical in response treatment predicting in aid could patients targets multiple from the cells in to choice of binding their and compounds of comparison interest. A of protein the on acts and tissue drug the intended the whether reaches showing reporter reliable a is tumors) (e.g., engagement target of measure the regimes they exert their effects. In a preclinical and clinical concentration setting, what at and setting relevant biologically a in gets tar of intended their engage questions actually candidates drug the fundamental whether the address to programs discovery drug in used be can CETSA curve, dose-response isothermal an as easily most but format, either In precipitates. and denatures protein unliganded the which at temperature the of knowledge a requires as latter The followed concentration. ligand be increasing can of function protein the of stabilization the which generated, in is curve dose-response isothermal an Alternatively, assessed. be can stabilization thermal potential a that such tures the in protein presence and absence of ligand is to subjected the a panel of tempera which in curves), aggregation ture-induced (or,tempera accurately, more curves melting apparent between engagement. target particular a firmly link the observed phenotypic response to a compound with to possibility the offers approach CETSA The function. target of ER Practically, the shift in is thermal stability estimated by measur comparison a is experiment CETSA a from output typical A K1/2. K1/2. ), P.N. ( 12,1 T he he assay involves treatment of cells with a [email protected] 3 . It is thus our hope that the CETSA method can be be can method CETSA the that hope .our thus It is 1 , Thomas Lundbäck S 2 Chemical Biology Consortium Sweden,Chemical Consortium Biology uch uch assays have been used ) ) or D.M.M. ( in vitro in R ecently, we published [email protected] and in specific tissues tissues specific in and 2 ,
T he he latter 9 . In this way, this In .
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© 2014 Nature America, Inc. All rights reserved. rarely rarely gives false CETSA positive data, ligand-binding in contrast to many approaches, TSA standard to similarly and experience, assay formats are difficult to establish for technical reasons. In our purify in a biologically active and relevant form or for which other be interest of for particular targets that are to difficult express and offer a broad range of screen assay alternatives, this approach may starting classes As many target programs. chemistry identify medicinal for to points serve could libraries compound sized ably assay. reporter attractive an be would method CETSA the in which applications of Hence, are a there drug. number the in for of patients whom selection the target remains responsive to kit diagnostics an of accompanying the basis ple constitute could and on impact the protein. to target Finally, princi the same underlying access confirm to studies clinical during cells patient in later also potentially and models animal in first tissues, different approach and reagents, one can next examine drug distribution in protein target the to availability ligand trol intact cells provide a versus means to study the that systematically aspects con lysates cell in engagement target of Comparisons aspects. protein-binding general and permeability example, for addressing, also thereby cells, intact chosen adequately in target the bind and reach to ability their of terms in characterized ther fur be then can molecules Identified CETSA. using lysates cell in target protein a defined of compounds for stabilizing libraries A drug could discovery program with a start screen of compound tems of increasing complexity and sys relevance to a clinical biological situation. of studies for platform technological common a binding. ligand demonstrate shifts substantial and compared, ( temperatures aggregation The antibodies. apparent obtained using target-specific detection blot western and electrophoresis gel target denaturing by, e.g., protein thermostabilized remaining the of detection finally and lysis, removal of cell debris and aggregates through centrifugation, the of cells to the and protein interest, denature of precipitate cell treatment of cells with either drugs or control, followed by heating CETSA process used in the proof-of-principle study starts with the The animals. in distribution drug as well as cells, mammalian in resistance drug and effects off-target activation, drug transport, drug efficacy, drug as such problems address to systems model of range by several a was using validated The targets. method drug different for information engagement target critical give can approach CETSA the such, As CETSA. using feasible is binding to owing ligand stabilization thermal of in cells, so measurement independently sufficiently precipitate and unfold often proteins publication proof-of-principle original our In the Applications method of applications. screening high-throughput and characterization low-throughput for protocols CETSA out p38 kinases human with tentative applications and their limitations. We have used the this along for in protocol the options available discuss and requirements methodology general the describe we Here challenge. heat a after protein soluble remaining of levels the evaluate cally specifi to ways several however, are, There proteins. isolated at CETSA is similar to assays used light scattering–based for looking activity-based assays activity-based For example, CETSA applying for screening of reason primary use to researchers allows it that is method the of feature key A α 14,1 and ERK1/2 as example cases for working working for cases example as ERK1/2 and 5 . Therefore, we see an immediate need need immediate an see we Therefore, . T agg ), with and without drugs, can be can drugs, without and ), with 1 . By using the same same the using By . 1 , we showed that that showed ,we ------
mechanism of action involving the target of interest. Along the the Along interest. of target the involving action desired of the mechanism to response phenotypic the link firmly to approach help will CETSA on-target the and assays close-to-target The assays. viability cell with example, for case, the is which targets, of often range a broad of may modulation of a as consequence across come response phenotypic desired the as engage crucial, is target ment specific a to coupling The readout. phenotypic engage ment to and response target in the functional the end also to the desired associating of purpose the serves This target protein). the of function presumed or known the on based functional assays (i.e., important cells an in as assays role ‘close-to-target’ a to have complement will setting cellular a in binding direct confirm to ability the We that protocols. believe detection generic enable to reporters or tags epitope include to option an provides also interest of target the overexpress recombinantly to or cells The possibility even tissues. cells, primary overexpressing lysates, cell in applications CETSA include also can target lated besides different of panel a in conducted be can compounds. experiments As such outlined, already confirmatory of set relevant most the on efforts downstream focus to order in targets isolated of campaigns screening high-throughput from for the verification of target binding for identified hit compounds only the folded protein). Below is a general description of two two of description general a is Below protein). folded the only recognizing on antibodies one based (e.g., entities these between material, or by using a detection method capable of distinguishing to the of samples remove or and precipitated denatured filtration either the of material, soluble e.g., achieved separation by through centrifugation be can latter The protocol). general the of line from proteins that denature and (see precipitate in which proteins step that a remain stable second, during heating and, are distinguished binding ligand through stabilized unless precipitate and denature proteins target which in step first, heating a components; crucial few a on relies protocol CETSA The design Experimental protein. target isolated active ligands and that would have been missed if assayed for with to become activation metabolic require that up compounds pick be can in cells are that CETSA able experiments ple to serves illustrate modification, intracellular synthase thymidylate of for inhibitors assessed as well as accumulation, the ligand. We have previously demonstrated that time-dependent of permeability cellular that control factors be of for used studies could proteins transport important overexpress or suppress that cells Alternatively, binding. protein plasma of effect the examine to culture cell the in concentration serum can varying by studied aspects be Such surroundings. its and cell viable intact the of reaches the target in in amounts sufficient a ligand complex background the that is response CETSA positive a for prerequisite A system. investigated the in ligand the of availability the affecting i.e., for aspects the target, affinity the intrinsic than other ligands studied the of aspects address help also will setting cellular a in screens. phenotypic in identified to compounds responds the proteome of portion sible (MS) spectrometry mass quantitative using i.e., purposes, identification target unbiased for approach CETSA the use to possibility the investigating are we lines, same The possibility to investigate target binding and stabilization stabilization and binding target investigate to possibility The T m shift assays and other biophysical studies on the iso the on studies biophysical other and assays shift natureprotocols 1 6 to study how the acces the how study to
| in vitro in VOL.9 NO.9VOL.9 1 . The latter exam protocol Fig. systems, which which systems, 1 | 2014 for an out |
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© 2014 Nature America, Inc. All rights reserved. gives a single band on the western blot for the protein of interest interest of protein the for blot western the on band a single gives ( experiment same lysates from several cell on lines using one screen or several quick antibodies in a the do to possible is it lines, cell immortalized in conducted be to are experiments If selected. be must protein get tar the toward directed antibody high-quality a protein, target for a given selectivity Second, to sufficient binding. obtain ligand allowing form and in a relevant quantities in sufficient expressed is protein the that is requirement the in minimal way. A biology possible best studied the reflects it that such chosen be must of the origin protein source (i.e., cell and type culture conditions) the First, important. are that choices and experiments initial few ligands. known of number limited a with approach is suitable for studies of one or a few proteins in parallel This degradation. protein target to prevent chosen appropriately are conditions assay that and antibody the by recognized indeed is protein correct the that insurance providing thus interest, of protein target the of size the of also but presence the of only not addition, In confirmation a provide visual interest. direct approaches blotting western of protein the toward directed specific a antibody is assay given a for required reagent unique only the and format, this in establish to simple laboratory are methods CETSA sis. biochemistry any as to western well blot systems running and for cameras analy quantitative as electrophoresis, gel available for equipment standard method housing a is It protein. soluble stabilized of detection the for choice attractive western dology using detection and blotting. preparation sample General of choice the method. to detection linked intimately is and variable also is to so, ways do the as well as material, precipitated and denatured the from protein stabilized remaining the of separation the as such used for cell lysis (when applicable). The need for sample workup, method the and heating sample for applied time and means the heating, before compounds with means treated is the sample the which extracts), by tissue or biopsies cells, intact lysate, (cell source protein the as such system, and protein target studied the many of the steps can be to varied of address aspects fundamental of known and anticipated limitations. As already alluded to above, outline an by followed protocol CETSA the of variants different analyzed accordingly. ITDRF, isothermal dose-response fingerprints. can be obtained by various means, and the soluble protein fraction can a be separation of soluble protein from cellular debris and precipitated proteinDepending on the detection method (i.e., western blot versus AlphaScreen), ITDRF obtain a CETSA melt curve or at a constant temperature in order to attainexposure an to the compound, samples are heated to various temperatures to alternatively, as a series of concentrations (for a ITDRF applicable, test compounds are added either as a high dose (for melta curve)CETSA or,melt curve or ITDRF collected from the various sources, they are aliquotted depending on whetherapplications are depicted on the right, in green. Once the samples haveprocedures been using samples of different origins. Envisioned future clinicalFigure 1 2102 be in tested that a order to few an antibodies appropriate validate recommend we and detection, quantitative allowing for ideal is protocol Before running CETSA on a new target of interest, there are a are there interest, of target new a on CETSA running Before
CETSA | VOL.9 NO.9VOL.9 1
| , we introduced a western blot–based procedure as an an as procedure blot–based western a introduced we , . After the heat challenge, the samples are homogenized and lysed. Schematic illustration of CETSA melt curve and ITDRF n u oiia peetto o te ES metho CETSA the of presentation original our In | 2014 Box Box CETSA |
1 natureprotocols ). Finding a high-quality antibody that that antibody high-quality a Finding ). experiment is designed. Similarly, when CETSA experiment). After CETSA
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of the cells must be optimized to ensure complete and uniform uniform and complete ensure to optimized be must cells the of protein fraction. In addition, the of cell method lysis after heating and from debris protein cellular the aggregates soluble remaining separating for means appropriate the and duration, heating ties, parameters are investigated, including sample volumes, cell densi other few a experiments, preliminary these During determined. stabilizing any of absence the apparent the that so ligands in established be must range) temperature wide a (spanning curve melting a cells, primary or protein. target the for cell conditions optimal about information valuable supply could to on find information expression profiles of the target of interest when also be chosen and carefully validated. In this regard, data mining peptides blocking of use applicable. the The appropriate cellular by condition or cell status or should bands the detected of on the analysis MS selectivity running by The validated further be this. can antibodies achieve to protocol blot western Before running CETSA on the protein in the selected cell line line cell selected the in protein the on CETSA running Before intact cells Lysate or In vitro cell debrisandprecipitate Centrifugation orfiltering from solubleprotein Collect organs of interest Ex viv Heat totemperaturerangeformeltcurve Separate Denaturing detection with compound Treat samples or selectedtemperatureforITDRF methods —SDS- + westernblot o Aliquot samplesformelting curve ordose-response PAGE Collect organs Treatment of interest T In viv Quantification +analysi agg lysis Cell curve Relative of the protein of interest can be be can interest of protein the of o
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© 2014 Nature America, Inc. All rights reserved. important given that ligand concentrations at the protein target target protein the at concentrations ligand that given important particularly is This the compound. in the as without used experiment control are cells of amounts similar that so carefully be monitored, should numbers cell and viability of terms in cells the on compound a of effect step. The preincubation the during to occur processes these for time enough to allow important is it Therefore, targets. intended their engaging before activated and modified are compounds certain Furthermore, membrane. cell the across compounds the of transport for time enough to allow crucial is it systems, cellular For exposure. of duration the and ligand the of concentration the as such considered, be must tors fac additional Here PROCEDURE). the of 1 (part inferred tion stabiliza of on extent the ligand information in to order obtain present known a with protein target the for curve melting a ing can place, be first by initiated, the CETSA experiments determin protein folded be of detection also direct by or can purification protein, affinity of use the by folded either envisioned the only affinity recognize of use that the reagents as such procedures, workup sample Other filtration. or centrifugation by achieved be can lysis and heating after fraction protein soluble and remaining the from debris aggregates cellular protein of removal The interest. of protein the of any aggregates solubilize not does detergent the that ensuring various using detergents), and (e.g., lysis protocols buffers with and/or without achieved systematically be can This kept is solution. protein in soluble the whereas samples, all in cells of lysis must exceed the dissociation constant to observe stabilization to observe constant dissociation the exceed must This image shows aschematic illustration of ahypothetic outcome from acellline and anantibody screen. Inthisparticular 9. Afterdevelopment and analysis, choose the antibody cellline pairthatgivesthe cleanest and sharpestsignals and bands. to the supplier’s recommendations. 8. Perform the SDS-PAGE separations and transfer the samplestonitrocellulose membranes; follow byantibody incubations according samples onmatching number of gels. 7. Loadeachcellline sampleonseparate lanes inapreferred gel. Depending onthe number of antibodies tobetested, loadthe same manufacturer’s recommendations. 6. Remove analiquot from eachcleared cellline lysateand mixwiththe SDS-PAGE loading bufferof yourchoice and follow the 5. Briefly vortex the tubes and centrifuge the tubes containing the cell lysates at 20,000 4. Freeze-thaw the cellstwice using liquid nitrogen and athermal cyclerorheating blocksetat25°C. 3. Add abufferof choice tothe tubes, resuspend the cellsand snap-freeze the cellsinliquid nitrogen. 2. Collectasuitableamount of eachcellline and washthe cellswithsuitablewashbuffer. 1. Expand the celllines tobescreened according toastandard cellculture protocol. P blots using yourantibodies of choice. ply runSDS-PAGE gels withanarray of celllines thatcouldbebiologically relevant for the experiment; follow thisbywestern To find and optimize the antibody and cell line used for CETSA western blot–based experiments, a simple screen can be conducted. Sim lines andantibodies experiment, antibody 3and cellline 2offer the bestpossiblecombination for westernblotexperiments. Box 1 When the basic components of the protocol have been put in in put been have protocol the of components basic the When rocedure | Antibodyandcelllinescreen 1 6 . 2 1 Antibody Cell line 4 3 1 6 5 2 1 2, Antibody 2 5 ● Cell line - - - , 4 3
T response curve increases with temperature in most cases, which which cases, most in temperature with increases curve response dose- the of point inflection the to reach required concentration ligand the that given possible, as low as kept is heat temperature the ing thumb, of rule a As °C). 37 often (most temperature relevant biologically the at those with compared dose-responses the at observed in or deviations minimize to maximal temperature is possible lowest difference the which at temperature a at points data to such a it work to may of difference, obtain be required preferable number the and experiment the of on purpose Depending the samples. control and ligand-stabilized in levels protein between observed is difference significant statistically a dose- can response experiments be at performed at temperatures which Such temperature. fixed a at experiments dose-response stabilization the doing involves PROCEDURE the 2 of potential part compounds, with including curves, aggregation temperature- induced establishes PROCEDURE the of 1 part Whereas PROCEDURE). dose- the of 2 isothermal (part run an be can which experiment at response temperature the visualize size, to shift is absolute the on information obtaining besides cells, control and cells in treated targets protein the for curves melting section. the in ‘Limitations’ detail more in discussed be will This lost. be could off-rates rapid with ligands and experiment, the in out, equilibria the affect pointed will steps be washing such however,that should It drug. excess any of cells the wash to optional also is it step, heating in the Before assays. applied cell those functional than higher the be to in likely used are approach CETSA concentrations compound the that means which IMI 6 5 The primary objective of the experiment comparing apparent apparent comparing experiment the of objective primary The N G 2 1 variable, dependingoncell Antibody Cell line 4 3 g for 20 min at 4 °C to pellet the cellular debris. 3 6 5 natureprotocols
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2014
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© 2014 Nature America, Inc. All rights reserved. (unless (unless detection can be made in the same plate). Finally, the detection is The diluted samples must then be transferred to a suitable detection plate debris and protein aggregates by means of centrifugation and/or filtration. are diluted with lysis buffer. At this point, it is optional to remove the cell temperature for 3 min. The plate is then allowed to cool before samples before placing the microplates in a PCR machine for heating to a predefined suspension to all wells. The samples are next preincubated for 30 min are first dispensed into individual wells followed by the addition of a cell Figure 2 and equipment, handling liquid automated using done be can additions reagent and sample all that means format This format. based screen-compatible equivalent)- (or microtiter fully a to steps a all of transfer that the requires clear is it steps, assay selected only for envisioned be can format throughput higher a to transition the Although throughput. sample daily higher edly points, some require the of a tentative CETSA applications mark detection. data hundred to a few up accommodate can format blot western homogeneous with format Screen compounds. identified the by engaged are targets which to respect with readouts screen typic pheno of deconvolution allow also possibly could approach this mentioned, already As effects. off-target of view comprehensive more a provide to and proteins of panel broad a of putative stabilization identify to used be could technologies MS-based tive, or As be could colored fluorescent. an which alterna themselves, compounds investigated the from or biotin medium culture and cell the in red phenol from interference as such formats, tion detec in other may arise that to it prone is artifacts proteins, less SDS-PAGE–separated of detection the on based is blotting ern and accurate interpretation. quantification Furthermore, as west and thus separation to the proteins sufficient allow between sizes molecular in differences substantial as well as protein, each for ever, requires highly specific antibodies giving rise to a single band how This, monitored. be must effects adverse which for targets (e.g., proteins related kinases such as cyclin-dependent kinases to homologous selectivity investigating when useful In compounds. this format, the this is of investigated part effects off-target and selectivity about information relevant provide and other applicable, against if antibodies proteins in the same western can blot increase experiment output addition, several In with exposed proteins. multiplexing to denatured antibodies in high-affinity epitopes of availability excellent concentration. ligand in increments desired with and from any concentrations can of be curve number established apparent the or below at conducted also are experiments cases certain in but aggregated, and denatured apparent the above temperatures at conducted are studies dose-response fingerprints or ITDRF dose-response isothermal as results these to refer we response, timing, as well as test temperatures, the influences dose- observed of experimental conditions, including cell medium conditions and under As section). the choice ‘Limitations’ on interpretation data see also the discussion in our laboratory; observations (empirical concentrations ligand of elevation into an unnecessary translates 2104 incubations and readings. achieved by following a standard protocol for AlphaScreen bead additions, protocol A major advantage of western blot–based detection is the the is detection blot–based western of advantage major A
| VOL.9 NO.9VOL.9
| The screen format assay procedure. Compound stock solutions T agg , at which a majority of the unliganded protein has has protein unliganded the of majority a , which at | 2014 CETSA | natureprotocols (ref. 1 ). In our laboratory, most of the T agg . The dose-response . dose-response The Whereas the the Whereas 1 ), or a few i cularly cularly ------
out any wash or sample workup steps. This assay procedure is is in procedure outlined assay This steps. workup sample or wash any out with assay homogeneous a on completely based is that protocol CETSA screening–amenable high-throughput a of example one on focuses below description the Instead, detail. in alternatives prior on these through go depending not will we setup, instrument and laboratories, experiences between vary will method assays (TR-FRET)-based transfer AlphaScreen as such structure, folded the recognize reagents affinity other or include in for methods detection which homogeneous antibodies alternatives Obvious lysate. cell as well as form, aggregated and in a denatured protein same the of a background against protein stabilized of quantification the allow hence should method ideal number assay of steps must be down brought to a minimum. The strategies MS targeted or different ELISA variants, proximity ligation assays can be a using assays, achieved available broad of range including fraction Subsequently, soluble the in protein target of steps). amount the of detection filtration and/or centrifugation by protein (i.e., aggregated and denatured the from protein soluble of plate– separation the microtiter to applies also using latter The equipment. achieved compatible be can cooling and heating a a smaller volume of compound solution, as this facilitates mixing. mend adding a large volume of a cell homogeneous tosuspension in outlined As homogenates. tissue or cells, suspension or adherent lysates, cell in performed is experiment cautions taken to achieve this may vary depending on whether the pre the and wells, respective the in material all to access com pound homogeneous ensure to is solved be must that Another cost. challenge and variability assay overall the reduce to plates between transfers sample minimize to practice screening good is it First, protocol. CETSA microtiter-based importance a developing of when issues additional of number a are there source, Besides considerations regarding the choice of protein protein of choice the regarding considerations Besides Read signals Centrifugation/ filtration Optional step Figure Figure 2 1 or time-resolved fluorescence resonance energy energy resonance fluorescence time-resolved or lysis Cell Incubate 2 . 2 0 . To further optimize throughput, the the throughput, . To optimize further to detectionplat of lysedsample Transfer aliquots in lysisbuffer samples Add donorbeads Compound dispenser Dilute 2 ECHO 2 s e . As the choice of detection detection of choice the As . Heat samples Figure Figure Incubate to compounds 1 Add cells 7 Add acceptor , dot blots Incubate 2 , we recom ,we beads 18,1 - - - - 9
© 2014 Nature America, Inc. All rights reserved. small sample volumes and high-density microtiter plates. As As plates. microtiter high-density and volumes sample small in pursued be to screening enabling miniaturization, to nability in outlined as candidates, antibody additional of testing allows that selection pair antibody for approach a similar the expanding CETSA toolbox to other targets effi cient selection of suitable antibody for pairs that can be applied when approaches An published of respectively. presence the bead, is benefit donor added streptavidin-coated a acceptor and A–conjugated bead protein a to antibodies the of binding that after is achieved target the protein of such recognition selective highly epitopes different two recognize that pairs antibody on based are assays SureFire The alternatives. these compare to work this of scope the outside is it but detection, homogeneous to achieve available options other several are above, there cussed commercial SureFire a using technology AlphaScreen the on fell approach CETSA the screen-format to attempts initial our levels, protein endogenous of detection the for assays well-validated with compatibility its which of case. individual each for necessity investigated carefully be must the the material, facilitate soluble to of included detection be selective can step separation centrifugation filtration optional or An proteins. between vary to it likely addition is in and unknown, largely is aggregates intracellular homogeneous the in a proteins unfolded for the of nature the prerequisite because is assay. This a is debris aggregated cell and and denatured proteins of background the against protein themselves. compounds from cell medium such components as phenol red, biotin and the detection the with interference reduce to serves also buffer lysis a of protein. volume of larger over target The addition denatured stabilized is remaining of detection the of selective no there perturbation that provided warranted are they formats, throughput high- with compatible more and faster are procedures lysis cell many plates a during campaign. screening As detergent-mediated dling become will step a in rate-limiting processing the of parallel han this cycles) freeze-thaw repeated (i.e., protocol, original the in procedure lysis the with compatible is format microplate a to transfer the a Although wells. between become variability of source otherwise major would this as lysis, cell complete of ment heating. after wells between rates cooling consistent ensure to used are cooling blocks Matched plate. each on assayed be to temperatures six at least enabling zones, heating separate with block a use heating a it to curve is ture, melting advisable when constructing whereas tempera single a requires only block heating the experiments, isothermal For heating. controlled carefully for machine PCR a considered. be must setups other that such biology relevant and expression target retain to support solid a require for most systems, but there may be cases where cell adherent lines acceptable be may This min. 30 of time incubation an and cells suspension of use the on based is case example shaken Our required. be if optionally can microplate the preincubation, the as to referred compounds, with incubation subsequent the During the sample volumes are decreased, there is the possibility to test test to possibility the is there decreased, are volumes sample the A A major assay advantage of homogeneous formats is their ame On the basis of available in instrumentation our and laboratory soluble and stabilized remaining the of detection Selective achieve the is performance assay optimal for prerequisite A to transferred is plate microtiter the preincubation, the After 2 3 kit against the protein kinase p38 2 4 . We are pursuing Box Box 2 . α . As dis ------
the PROCEDURE section for intracellular human p38 human in for intracellular section detail the PROCEDURE in described are format detection AlphaScreen-based an and blot– western a both using experiments CETSA forming procedures. CETSA Example section). (see ‘Limitations’ equilibria of and on the minimize heating of throughput effect rearrangement increase further thereby and times heating reduced substantially by ITDRF by followed are experiments curve melting below. The discussed as systems, cellular and types cell other accommodate to necessary be may protocol the to Variations inhibitors. known of presence and absence the in °C 67 to 40 from spanning temperatures, ent p38 soluble measured on based is curve ing melt The candidates. drug stabilizing of presence and absence in the of target the curve melting the of apparent an examination with order starting interest, of target protein new a chronological approaching when recommended the in outlined are herein in found be can format screen a in workflow CETSA practical the of illustration in illustrated schematically are means the two detection assays, including these of workflows overall The cells). HL-60 intact on performed are experiments In complex systems such In as or systems cells complex cell lysates, it may be also that forms. various the between distinguish cannot reagents affinity the and dominating are forms nonfunctional the particu if helpful be larly could methods MS-based to Moving addressed. is methodology detection and/or antibody of choice the which in experiments troubleshooting appropriate by followed be result negative a that crucial is it reason, this For processing). teolytic pro or the necessary phosphorylations activating a lacking form (for the example, protein of form a of nonfunctional recognition the lack of observed stabilization cases, may be the consequence certain of antibody In established. well been yet not have respect this in assay the of limitations the although assays, shift thermal in response ligand-induced no or weaker give to likely more are complexes, protein including proteins, Larger environment. lar cellu a in assays shift thermal performing when binding also issue an is ligand upon observed not is stabilization protein that sense the in negatives false of occurrence the TSAs, classical the for as rare, are positives false Although methods). MS-based tive reagents, of affinity can independent be employed (e.g., quantita reagents are which affinity and on techniques, that the fact purposes for detection suitable of availability the on dependent also is It protein. target the of stabilization substantial a induces and a lig of binding the whether on dependent is ligand bound with protein a for curve melting CETSA a establish to possibility The and Limitations other considerations p38 to regard with activity of unknown of samples screening primary CETSA-based performing for procedure a provide also we RESULTS,ANTICIPATED the in outlined As °C. 50 at performed is it example this in and compound), lizing stabi of absence the in detected not and precipitated is protein the of majority a which at temperature a at (e.g., curve melting ITDRF the which at temperature The step. heating the during perature pound is concentration with varied 11 doses tested at a fixed tem com the that except experiments, curve melting apparent to the CETSA CETSA experiment is performed is based on the observed observed the on based is performed is experiment experiments in the same cells. These are similar similar are These cells. same the in experiments Figure Figure natureprotocols 2 . The types of experiments included included experiments of types The . Figure Figure Experimental protocols for per for protocols Experimental 1 , whereas a more detailed detailed more a whereas ,
| VOL.9 NO.9VOL.9 α levels at ten differ ten at levels protocol α stabilization. | 2014 α (i.e., the (i.e., |
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© 2014 Nature America, Inc. All rights reserved. all equilibria all involved;equilibria for example, the binding to serum proteins target longer residence times have that or cells the in retained are com that for the pounds bias no in is there that compounds ensures heating Leaving during medium curves. dose-response or melting curves apparent generate to heated are cells the which under methodologies. detection other or antibody the of choice the to addition in system, cellular the of choice ate appropri the on dependent is stabilization ligand-induced a of allosteric for In binding. regulators proper ligand this case, the or observation partners interaction requires interest of target the 2106 demon strated already As affected. be also may permeability cell and protocol sign of target engagement. is testedatatemperature wellbelowthe apparent is advisedtousealternative detection methodologies. Alternatively, counter-screening canbeconsidered, inwhich signal quenching suitable pair, recognizing allentities of the protein target similarly(e.g., unliganded and ligand-bound), cannot beidentified, the user Data specific tothe protein of interest canbeeffectivelyscreened. anti-FC domain antibodies toward antibodies generated indifferent species, alarge portion of commercially availableantibodies valuable when screening for antibodies, asitcaninfluence the strength of the AlphaScreen signal. Byusing abattery of capturing of the respective antibodies oneither acceptor ordonor beadscanbevaried bychanging the beadfunctionalization. Thisis a directly conjugatedsecondary antibody thatrecognizes the Fcdomain of anantibody toward the target of interest. The positioning selectivity onseparate beads, alarge proportion of availableantibodies canbetested. dual protein Aaffinityapproach, aswellapproaches thatinvolvetwoanti-Fc domain–directed antibodies withdifferent species which isdirectly conjugatedtoeither acceptorordonor beads(the latterisillustrated below),isspecies-specific. Together withthe primary antibody withpoorprotein Aaffinity. Thisapproach adds additional specificityto the system,as the capturing antibody, one beadwithacapturing antibody onthe other bead. Thissecondary capturing antibody isdirected toward the Fcdomain of a kinase). Analternative means toextend the range of antibodies availablefor testing istocombine the protein Afunctionality on biotinylation, such thatthey caninstead becaptured onstreptavidin-functionalized donor beads(applied inthisworkfor the p38 antibodies asdefined byspecies and isoform of availableantibodies for target protein recognition (becauseprotein Aonlyprovides sufficient high-affinity binding tocertain risk of antibody rearrangements betweenbeads, which may influence signal levels, aswellthe exclusion of asignificant portion functionality onboththe acceptorand the donor AlphaScreen beads but identification of anappropriate antibody paircanalsobeachieved inwell-designed screening approaches. which antibodies totestare preferably made onthe basisof availableinformation onepitoperecognition and target protein structure, protein exposes additional epitopes(i.e., ispartially unfolded), such thatthe choice of antibodies isbroadened. Choices regarding detergents, aswellbufferpHand ionic strength, inwhich the antibody pairisfullyfunctional butinwhich the solubletarget cannot beachieved, itisoptional (aside from changing detection methodology) tosearch for conditions, e.g., levelsand identity of exposed inthe native protein, ascompletelydenaturing conditions are not compatible withantibody functionality. When this affinity and selectivity for the target protein. Importantly, this must beachieved onthe basis of target epitopesthatare well A prerequisite for homogeneous antibody-based detection assaysisthe identification of asuitableantibody pairwithsufficient Box 2 A matter of great importance is also the choice of conditions conditions of choice the also is importance great of matter A On the basisof ourexperience withligand-induced quenching of the antibody pairrecognition (asdemonstrated in Shown aboveisanillustration of one example of the combined useof ahigh-affinity protein A–binding antibody together with As outlined inthe main text, there are published approaches for efficient selection of suitableantibody pairswithprotein A
| VOL.9 NO.9VOL.9 ), werecommend thatthe antibody selection beperformed bothinthe presence and absence of asetof known ligands. Ifa 1 , the cells are left relatively intact during the transient transient the during intact relatively left are cells the , | Genericformatforantibodypairselection 2 5 | . However, may heating result in of an alteration 2014 | natureprotocols Donor bead e.g., Anti-mouseIgG 3 Matching thecapturingantibody, 2 Capturing antibody ). Efficient screening of antibodies withpoorprotein Aaffinitycanbeachieved by e.g., raisedinmouse protein with low affinity Target antibody T agg A of the target protein, assubstantial signal alterations inthemselves are a 1 - - - 2 4 have also pursued screen-compatible methods with the potential the potential with methods screen-compatible have pursued also This is a key reason why we, in to parallel the western blot format, to and device heating the improve geometry sample heat transfer. to or volume alter to reduce the sample it completely is necessary scale, minute than rather second the on times Toheating obtain volume. sample with increases sample complete the in neously homoge so do to and temperature certain a time reach to the required microplates, or vials sample the for blocks heating of types or other cyclers thermal When using cooling. and ramping temperature uniform and efficient requires which time, the heating reduce to is equilibria the of rearrangements of impact the and for system. each ligand studied A taken to measure minimize be must equilibria these in changes potential of kinetics the but °C ( 60–65 of up to temperatures heating . Although fullyfunctional, thisapproach isassociated withthe Target protein e.g., Rabbitderived to protein Antibody binding A Acceptor bead Protein coated A Supplementary Fig. 1 Supplementary S upplementary
α
), -
© 2014 Nature America, Inc. All rights reserved. from folded to unfolded protein. This is an important argument argument important an is This protein. unfolded to folded from condition controls the extent to which aggregation drives the shift time in as heating the with CETSA this experiment, experimental investigated in detail, the user can expect these parameters to vary transitions irreversible for curve ( midpoint apparent the and shape the both determining in role key a have will protein unfolded the of tion in aggrega involved kinetics the transitions, two-state reversible for curve melting the of shape the determines which protein, the irreversible two-state a by model approximated be sometimes can (N protein to aggregated unfolded from through native transition the aggregation, and unfolding protein of kinetics the experiments curve melting in aggregated and in proteins which isolated are denatured experiments irreversibly T correlation has been demonstrated between observed equilibrium cells. within protein However,denatured precipitates an excellent transitions at non-equilibrium, such as those we observe when the protein unliganded the of change capacity heat and enthalpy unfolding the of mination deter experimental independent require calculations The shift. the of dependence concentration the ligand of the knowledge of basis the on determined quantitatively be can affinities ligand transition, two-state a reversible with comply that proteins lated fully from native to (i.e., completely denatured and material). aggregated For iso environment complex its in protein target the of denaturation thermal the describe accurately that models ing requires establish to affinities, ligand fingerprints dose-response terms of converting the apparent in responses, CETSA observed the of interpretation quantitative range of different target proteins in our laboratory. broad a However,for ligands between a ranking relative firm and binding of tion ments have already been proven to be valuable for the demonstra times. longer require to known events cellular specific address to is intention the unless min, 30–60 than longer times preincubation involve not do generally protocols our reason, this For activities. lation trans and transcription altered through levels protein influence also could compounds hours, toward extend the times If time. incubation preincubation the on dependent be will responses such and specifically, protein target the for or activity, general in proteasomal either altered as such levels, protein affect that responses functional induce also may compounds the Similarly, quantification. blot–mediated western before forms different of separation the facilitate to used be could electrophoresis gel 2D Alternatively, form. either detect that methods MS-based use to or antibodies forms protein use different the to between discriminate preferable not do that be will it cases, these In protein. the detect to ability antibodies’ the influence may turn in which pathways, it protein if is signaling involved in responding quickly target the of status post-translational the may effect compounds an the in is because This protein. target the result to recognize ability altered could this as compounds, the with preincubation volumes. microliter single-digit and format for extreme miniaturization, possibly even down to the 1,536-well for minimizing the heating times, besides the influence on cell cell on influence the besides times, heating the minimizing for m The ITDRF The for used time the to relates aspect kinetic important Another values and apparent apparent and values 2 6 . Besides the unfolding enthalpy and heat capacity of of capacity heat and enthalpy unfolding the Besides . CETSA values obtained from dose-response experi dose-response from obtained values 2, T 5 agg . These relationships do not apply to to apply not do relationships These . values derived from light-scattering light-scattering from derived values T 2 6 agg . Although this remains to be be to remains this .Although values, as well as isothermal T agg 9 . Depending on on Depending . ) of the melting melting the of ) → U → T A) A) m ------
a thermal and detergent-mediated denaturation of all soluble soluble all of denaturation detergent-mediated and aggre thermal a not involves format but blot western the unfolded because is This protein. partially gated native also possibly unliganded and and protein ligand-bound i.e., of protein, the target subpopulations that the several of indicate consists may fraction it protein formats, soluble assay between observed is affinities. ligand and responses observed between relationships quantitative the them with and proteins, target of and aggregation denaturation the intracellular describe better that models of for derivation the data these apply to and proteins target different of range broad a on data mulate accu to is goal long-term Our cases). most in temperature increasing test with concentrations curve ligand higher dose-response toward the increases of point inflection the (i.e., proteins target isolated from observations with agreement qualitative in ITDRF proteins individual the for aggregation and denaturation thermal independent an with compliance reasonable in are far so observations empirical our surprisingly but environment, cellular complex a in occurs tion aggrega the experiments, CETSA the In this. achieve help will membrane integrity, and further minimization of sample volumes bilayer (e.g., interfacial proteins, or extracellular or intracellular intracellular or extracellular or proteins, interfacial (e.g., bilayer However, that protein are for domains membrane not in the lipid CETSA protocols for monitoring ligand binding to such domains. establish to challenging be might it thus and temperatures, high very at unfold therefore and bilayer lipid by the stabilized highly in cells are domains intact often CETSA membrane data. Integral generate to potential the has also lysate a in proteins membrane transporter membrane as CETSA have been applied principles to monitor same ligand binding the to an integral using proteins membrane gent-purified interfacial of deter on assays shift subsets Thermal proteins. for membrane integral also and well work will method the that proteins soluble involved only CETSA of study pilot the Although addressed. be can proteins target which also is ods protein. stabilized the the of recognize to ability antibodies the quench ligands the if situation the in handle to described is selection pair antibody for strategy a noted, already As binding. ligand upon unaltered remains recognition which for pair antibody alternative an for in below shown searching the ANTICIPATED we recommend RESULTSis section, which of example an target differentially, unliganded protein and ligand-bound homogene detects the format in assay used ous pair antibody the if Similarly, not. or ensure it native whether protein, is of stabilized ligand-bound regardless to of format subpopulation blot intended the measures western protocol final the that the from results with comparative unliganded by studies and addressed be only can ligand-bound matters These between protein. ratio the will dilutions influence extensive and detergents as protocols, handling sample cell lysis and protocols different the downstream between recog vary may turn, in This, pairs. differently antibody or are antibody the by nized these if subpopulations differently different respond the may to denaturation that prior sub format involve not assay all any does of whereas behavior detection, same during the populations expect we thus and material, When a discrepancy in apparent apparent in discrepancy a When Of Of importance for the general applicability of the CETSA meth CETSA temperature influences the observed stabilization stabilization observed the influences temperature natureprotocols 2 1 7 9 . Direct heating of detergent-solubilized . detergent-solubilized of heating Direct . We further observe that the choice of of choice the that observe .We further T agg or responses to ligands ligands to responses or
| VOL.9 NO.9VOL.9 Box Box 2 protocol , including how how including , | 1 2014 , it is likely likely is ,it |
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© 2014 Nature America, Inc. All rights reserved. • • • • • • • • • • • • • • EQUIPMENT • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • REAGENTS M in used be can procedure step–based separation a Alternatively, that antibodies recognize the folded structure (e.g., AlphaScreen). probably be done with the above,discussed direct strategies using can This generated. be principle in can curves melting CETSA-type apparent and proteins, soluble for observed that to similar be will unfolding the proteins), membrane integral of domains 2108 • protocol (5–300 Electronic (Optifit pipette tips Tips, cat. nos. 790010(0.2–10 (2–200 Pipette epTips (epTips, cat. nos. 05-403-39(0.2–20 Thermowell aluminum (Corning, sealingtape cat. no. 6570) CyBi-TipTray 9625 cat. nos. 730320(0.2–10 eLINE;Electronic multichannel eight-channel, pipettes (Sartorius (Rainin,Pipette tips SS-L10, SS-L250andSS-L1000) and L-1000XLS) channel pipettes (Rainin,Single Pipette-lite L-2XLS, L-20XLS, L-200XLS PowerPac basicpower supply (Bio-Rad, cat. no. 164-5050) (Life Technologies, device iBlot geltransfer cat. no. IB1001EU) XCell4 SureLock Midi-Cell (Life Technologies, cat. no. WR0100) Leica DMILinverted microscope (Leica Microsystems) INCO 108CO MicroAmp (Life Technologies, strip eight-cap cat. no. N8010535) MicroAmp strip, eight-tube 0.2ml(Life Technologies, cat. no. N8010580) Veriti cycler 96-wellthermal (Life Technologies, cat. no. 4375786) AlphaScreen IgG detection kit(Protein A; PerkinElmer, cat. no. 6760617M) AlphaScreen SureFire ERK1/2total (PerkinElmer, cat. no. TGRTES10K) TGRT38S500) AlphaScreen SureFire p38MAPK cat. no. 170-5061) western enhancedClarity chemiluminescence (Bio-Rad, (ECL)substrate Biotechnology, cat. no. sc-2055) HRP-conjugated anti-mouse Goat Cruz (Santa antibody secondary Cruz (Santa Biotechnology,antibody cat. no. sc-2374) peroxidase horseradish anti-rabbit Bovine (HRP)-conjugated secondary cat. no. sc-69879) Anti– cat. no. sc-535) Anti-p38 cat. no. IB3010-01) stacks,iBlot transfer nitrocellulose, regular size (Life Technologies, NuPAGE MES SDS running buffer (20×; Life Technologies, cat. no. NP0002) (Life Technologies, cat. no. WG1403BOX) NuPAGE Novex bis-Tris 4–12%(wt/vol) midigels, 26well (Life Technologies, cat. no. LC5925) SeeBlue Plus2 prestained protein standard weight molecular NuPAGE reducing agent (10×; Life Technologies, cat. no. NP0009) NuPAGE LDSsamplebuffer (4×; Life Technologies, cat. no. NP0007) Trypan blue (0.4%(wt/vol); Bio-Rad, cat. no. 145-0021) l FBS (Life Technologies, cat. no. 10500-064) Antibiotic-antimycotic solution (100×; Life Technologies, cat. no. 15240-062) RPMI-1640 medium (Sigma-Aldrich, cat. no. R8758) HL-60 cell line(ATCC, cat. no. CCL-240) cOmplete, EDTA-free protease inhibitors (Roche, cat. no. 05056489001) Nonfat milkpowder dry (Semper) Tocris,ERK 11e(VX-11e; cat. no. 4465) SB203580 (Tocris, cat. no. 1202) AMG-548 (Tocris, cat. no. 3920) DMSO (99.9%; Sigma-Aldrich, cat. no. D8418) Kinase buffer (10×; Signaling, Cell cat. no. 9802) TBS-Tween (TBST; tablets Calbiochem, cat. no. 524753) (PBS, pH~7.4(Sigma-Aldrich, cat. no. P4417) Liquid nitrogen (useany provider) local ATER -Glutamine (100×; Sigma-Aldrich, cat. no. G7513)
| β VOL.9 NO.9VOL.9 -actin mouse primary antibody (Santa Cruz (Santa Biotechnology, antibody mouseprimary -actin I µ µ ALS l) and791200(50–1,200 l) and05-403-68(50–1,000 α rabbit primary antibody (Santa Cruz (Santa Biotechnology, antibody primary rabbit 2 incubator (Memmert, Fisher Scientificcat. no. 11574306) | 2014 µ l (CyBio, cat. no. OL3800-25-533-N) µ l), 730340 (5–120 | natureprotocols µ α l)) Total (PerkinElmer, cat. no. µ l)) µ l) and730390(50–1,200 µ l), 05-403-41
µ l), 790302
µ l)) • • • • • • • • • • solubilized. not are aggregates that ensuring while protein(s) target the solubilize efficiently to detergents different of screening including optimization, stantial sub need to however,likely is, procedure latter The step. ration sepa centrifugation-based a before just done is lysate cell the in proteins membrane the of solubilization detergent mild a which protease inhibitor to thePBSbefore use. 12–24 months atroom temperature (20°C). Add complete EDTA-free water, ultrapure of autoclave itandallow itto cool. life is shelf The PBS the same. than insuspension, asoutlined herein, thedownstream steps with remaining incubation step inthecell culturebefore flasks detaching the cells Such comparative achieved studies canbe by thecompound performing compared presented theformat with here (i.e., detached andinsuspension). any differences engagement incells indrug-target growing asamonolayer would,validation for example, involve theinvestigation there whether are of this protocol must carefully be validated for each system. individual This growingcells asmonolayer normally cultures, in their useasdescribed and HL-60 cells are used in the example used in the procedure. For adherent Cells 2 of mM before seeding and cells splitting in culture flasks. culture experiments. Fresh Preheat the culture medium to 37 °C using a water bath before use in cell medium at 4 °C. supplemented The shelf-life of medium is ~4–6 weeks. streptomycin and 250 ng/ml Fungizone. Store the supplemented cell culture solution to a working 100 concentration of units/ml penicillin, 100 ing FBS to a final 10% concentration of (vol/vol) and antibiotic-antimycotic Cell culture medium REAGENT SETUP • • • • • • • • • • • • • • • • Moxi Zautomated cell counter (VWR, cat. no. 734-2477) Costar 12-wellculture plate (Corning, cat. no. 3512) and T175, cat. no. 353112) cultureCell flasks(BD Falcon; T25, cat. no. 353109; T75, cat. no. 353136; cat. no. 86.1253.001; and10ml, cat. no. 86.1254.001) Serological pipettes (Sarstedt, 2ml, cat. no. 86.1252.001; 5ml, cat. no. 62.547.254) Conical (Sarstedt, tubes 15mlcat. no. 62.554.502; 50ml See-saw rocker SSL4(Stuart, SSL4) Milli-Q system (Millipore) ChemiDoc MPsystem (Bio-Rad, cat. no. 170-8280) 72.695.500) Microcentrifuge (Sarstedt, tubes 1.5mlcat. no. 72.706; 2mlcat. no. cat. no. 5424708.005) M cat. no. LP-0200) Echo qualified 384-w Echo 550liquid (Labcyte) handler cat. no. 6008289) ProxiPlate-384 Plus, white shallow-well microplate (PerkinElmer, Twin.tec PCR96-wellplate, (Eppendorf, skirted cat. no. 0030128672) Nunc U-bottom 96-wellpolypropylene plates, clear (Nunc, cat. no. 267245) TECHNE TC-PLUS cycler thermal (Bibby Scientific, cat. no. ELITE02) IKA-Schuttler MTS 4microplate shaker (IKA) CyBi-Well 96/384-channel simultaneous pipettor (CyBio, cat. no. 3391 3 4112) cat. no. 24073290) Scientific, cassette dispensing tip (Thermo plastic Small tube Multidrop Combi Scientific, reagent dispenser (Thermo cat. no. 5840300) Scientific,Standard cassette dispensing tube (Thermo cat. no. 24072670) Multidrop Scientific, 384reagent dispenser (Thermo cat. no. 5840150) Envision reader 2104multilabel (PerkinElmer, cat. no. 2104-0010) Counting slides for TC20 (Bio-Rad, cat. no. 145-0015) TC20 automated cell counter (Bio-Rad, cat. no. 145-0102) Moxi ZType Scassettes (VWR, cat. no. 734-2482) icrocentrifuge (Eppendorf, cat. no. 5424000.215; PCR-tube rotor,
Prepare PBSbuffer by one in 200ml PBStablet dissolving 200mlof This protocolThis cells insuspension, optimized hasbeen for usewith
ell lowell dead volume microplate (384LDV; Labcyte, Supplement the RPMI cell culture medium by add l -glutamine is -glutamine added to a working concentration
1 rather rather
µ g/ml g/ml
-
- - © 2014 Nature America, Inc. All rights reserved. detachment (using the method of yourchoice) ordirectly inthe culture containers. in suitablecellculture flasksormicroplates, withthe heating step(Step11)performed either immediately aftercell adherent cellsand itisdesirable toavoid detaching the cells, the compound incubation stepcould instead beperformed 2| experiment should beperformed atleastthree times, eachondifferent days, inorder toget statistically meaningful results. procedures and supplies. Approximately 120million HL-60cellsare required toestablishfour CETSA melting curves. This 1| plus cellstreated withDMSO alone asanegative control. different temperatures withendpoints spanning from 40–67°Cinthe presence of 20 p38 compound treatment P PROCE to uselowernecessary thecompounds concentrations inDMSOowing of dissolved inwater instead. For compounds, nonpolarorganic be it might skinpermeable.highly DMSOsolutions, handling when important asthesolventparticularly is potentially toxic workingwhen with andmutagenic compounds. is This ! are diluted to 4mMinDMSObefore useintheITDRF stored frozen as10mMstock solutions use. until 10mMstock The solutions 10mMstockin DMSOto solutions. yield compounds are our library All Compound preparation Excess discarded. mixshouldbe specifications.The lysis buffer immediatelybe used should forbest results. diluted fivefold water in double-distilled according to themanufacturer’s PerkinElmer buffer lysis insubdued light. performed immediatelyused for results. best Excess discarded. mixshouldbe according to themanufacturer’s specifications.The donorbe mixshould 20-fold indilution buffer (from theSureFire kit)immediately before use mix Donor bead can re-dissolve. re-equilibrate atroom temperature before usesothatthebuffer components bufferthe activation itisstored when intherefrigerator; itmust therefore Excess discarded. mixshouldbe immediately used mixshouldbe afterbead preparation for results. best the already andreaction activation prepared buffer. mixof acceptor The (frombeads theProtein A IgG detection kit)are next diluted 50-fold in SureFire kit)fivefold in bufferreaction (from the SureFire kit).acceptorThe manufacturer’s specifications by dilutingactivation the buffer (from the Acceptor mix bead solutionsubstrate for isneeded four midi-size nitrocellulose membranes. thewesterndevelopment blot membrane(s). of Approximately 12mlof peroxide andClarity substrate solution. Make fresh before ECLsubstrate kit ECLsubstrate western Clarity freshlybe prepared. TBSTto nonfat 5%(wt/vol) milk. obtain of blocking The buffer should Western blot blocking buffer life is1week atroom temperature. Milli-Q water Tween 0.05%(wt/vol) to TBSwith obtain shelf The (TBST). Western blot washbuffer 10× NuPAGE reducing agent before use. buffer by mixing560 Reducing buffer loading
art 1,determinationof theapparent meltingcurve for anintracellular protein by CAUT
CR
CR α I Add 15mlof the 2million HL-60cellspermilliliter of suspension into four separate T75flasks. Ifyouare working with Expand HL-60cellsincellculture medium toacelldensity of ~2million cellspermlusing standard sterilecellculture T (i.e., the experiment isperformed onintact HL-60cells).The curveisbasedonmeasured solubleprotein levelsatten I I I ON T CAL D I URE CAL Use appropriate equipment andacontrolled safety environment The donor The are beads light-sensitive, sowork must be Part 1of the PROCEDURE(Steps1–17)describes how toestablishaCETSA melting curvefor intracellular
Dilute donor (from beads theProtein A IgG detection kit)
The acceptorThe mixisprepared bead according to the µ l of 4×NuPAGEl of 240 LDSsamplebuffer with
CR
Compounds delivered aspowders are dissolved For 40samples, prepare 800 The 5×lysisThe buffer (from theSureFire kit)is Dissolve one TBS-Tween in500mlof tablet I ● T
I Dissolve 2.5 g of nonfat dry milkin50ml nonfat dry Dissolve 2.5gof
CAL T IMI
CR Water-soluble compounds canbe Mix equal volumes of Clarity luminol Mix Clarity equal volumes of I N T I G CAL 2–3h A precipitate may in form µ CETSA l of reducing loading l of experiments.
µ l of l of
INCO INCO 108 CO EQUIPMENT SETUP thecompound tomuch add. of thecells thatyouDMSO tolerance are before of working with deciding how to their lower solubility. thisisthecase, to If determine the itisimportant hence it will not affect thetotal notaffect significantly. incubationtime hence itwill This thermal must present be temperature during ramping, but thisisachieved in5s, and 50 °Cfor speed. 3minusingmaximum ramping In thisinstrument, theplate one-step intheTECHNETC-PLUS program cycler thermal for at heating TC-PLUS cycler block. thermal a96-wellheating with isequipped Create a cycler thecells ina96-wellplate format. for heattreatment of TECHNE The TECHNE TC-PLUS cycler thermal program. option chosen canbe design intheexperimental heat exposure, cycler; thethermal this to heatthelidof itisnotnecessary between thetubes two steps. heating for of handling the3-min During introduced thesametemperature asinthefirst with stage to allow moretime 58–73°Cfor 3min.stage spantemperatures of An intermediate step canbe the first stage spans temperatures from 40–55°C for 3minandthesecond in the Veriti cycler thermal temperature 3°C, increments with of inwhich theothertemperature zones. independently of Create atwo-stage program VeriFlex maintainingaspecific zones, 16wellscapable of each zone with of block96-well heating forinto divided 0.2-mlPCRtubes sixdifferent thecells.heat treatment of The Veriti cycler thermal a with isequipped Veriti cycler 96-wellthermal and 5% CO water in the incubator, and then set the incubator to 37 °C, 95% humidity ProxiPlate. the Twin.tec a384-well 96-wellPCRplate to within quadrant aselected in each step. In thesecond step, 4 stepsdispensing intheTwin.tec 96-wellPCRplate 25 usingavolume of two mainsteps.with and first sixsequential The step aspiration consists of a Twin.tec 96-wellPCRplate to a384-wellProxiPlate. Create aprotocol lysate96-channel from simultaneous pipettor of for mixingandtransfer CyBi-Well 96-channel simultaneous pipettor a Twin.tec 96-wellPCRplate. compoundsstock from 384LDV solutions of aLabcyte source plate to Echo Labcyte 550 msandexcitation 180ms(mirror time D640as). 570 nm, 100nm, bandwidth 75%, transmittance total measurement time AlphaScreen standard protocol emission settings: filter center wavelength at reader multilabel Envision Echo. theLabcyte with dispensing cycler theTwin.tec iscompatible with 96-wellplates for used skirted 2 .
2 Use to DMSO Echo 550instrument transfer theLabcyte incubator
µ Place Place a water tray containing sterilized ultrapure natureprotocols
M AMG-548,SB203580orERK11e Use theEnvision plate readerthe with
CETSA Use the Veriti cycler 96-wellthermal for µ l of themixed lysatel of from istransferred
Use theTECHNETC-PLUS thermal : cellhandlingand
Use theCyBi-Well
| VOL.9 NO.9VOL.9 protocol |
2014
|
2109 µ
l
© 2014 Nature America, Inc. All rights reserved. ensure consistent timing between tubes in both the heating and cooling steps. cycler. Only place the tubes in the blocks when the temperature has reached the designated temperature. It is crucial to Veriti 96-wellthermal cycler. Immediately afterheating, remove and incubate the tubesatroom temperature for 3min. 12| ? ensure consistent timing between tubes in both the heating and cooling steps, as well as between heat stages. cycler. Only place the tubes in the blocks when the temperature has reached the designated temperature. It is crucial to 3 min.Afterthis3-minincubation, immediately snap-freeze the samplesaccording tothe instructions inStep13. in the Veriti 96-wellthermal cycler. Immediately afterheating, remove and incubate the tubesatroom temperature for 11| Heat treatment of cell suspensions heat treatment step. (this setupfacilitates the tubehandling during heat treatment). The tubesare kept atroom temperature before the designated temperature (40–67°C).Thisyields atotal of 40PCRtubesdivided into tenstripswithfour tubesperstrip PCR tubeswith100 10| such interference can be tested in prior experiments for each individual target protein. ! cell pellet. 9| ? the cellsagain.Carefully remove and discard allof the supernatant (repeat thisstepifnecessary). 8| more sensitive to disruption during centrifugation or are more difficult to pellet. discard allof the culture medium. 7| ? of the compound-treated cells. 6| 5| ? 4| skin-permeable. and mutagenic compounds. This is particularly important when handling DMSO solutions, as the solvent is highly ! cells are much more sensitive, so it is advisable to do a pilot experiment to determine DMSO tolerance. cells in question is not exceeded. As a rule of thumb, avoid DMSO concentrations above 1% (vol/vol), but note that many serological pipette. as the vehicle orsolvent control. Gently mixthe cellsuspension bypipetting upand down several times using a get afinal concentration of 20 3| 2110 ? protocol
CAUT CAUT TROU TROU TROU TROU TROU
CR CR CR CR CR Inthe meantime, heat the remaining four stripsattheir designated temperature (58–67°C)for 3mininthe Heat the PCR-tubestripswiththe firstsixtemperature endpoints (40–55°C)attheir designated temperature for 3min Distributeeachcellsuspension, i.e., withDMSO control orwiththe testcompound, into tendifferent 0.2-ml
Add 1mlof PBSsupplemented withprotease inhibitors toeachrespective tube and carefully resuspend the Gently resuspend the cellpelletswith15mlof PBSand centrifuge them at300 Centrifuge the conical tubesat300 Count the cellnumbers and assesscellviability using apreferred method. Collect the cellsuspension withaserological pipetteand transfer the cellstomarked 15-mlconical tubes. Incubate the cellculture flasks for 1hinthe CO Add 30 | I I I I I VOL.9 NO.9VOL.9 T T T T T I I I I I I I B B B B B ON ON CAL CAL CAL CAL CAL LES LES LES LES LES Protease inhibitors must be avoided if they interfere with the target or system being evaluated. The extent of Use appropriate safety equipment and a controlled environment when working with potentially toxic
µ H H H H H STEP STEP STEP STEP STEP l eachof the 10mMDMSO stocksolutions of AMG-548,SB203580and ERK11etoindividual flasksto OOT OOT OOT OOT OOT | 2014 Do not let the temperature in the blocks rise to the designated temperature while the tubes are in the Do not let the temperature in the blocks rise to the designated temperature while the tubes are in the The centrifugal force may need to be adjusted if the cellular test system is altered such that the cells are It is important to examine whether the compound has acutely affected the viability or membrane integrity If the compound concentrations are adjusted, ensure that the predetermined DMSO tolerability of the I I I I I N N N N N µ G G G G G l of cellsuspension ineachtube(~3million cellspertube).Mark eachtubeorstripwitha | natureprotocols µ M of eachcompound. Add the same volume of DMSO tothe remaining flaskserving ●
g T for 3minatroom temperature topelletthe cells, and then carefully remove and IMI N G 10 min 2 incubator at37°C. g for 3minatroom temperature topellet
© 2014 Nature America, Inc. All rights reserved. ( points perday. whereas the AlphaScreen format canbeapplied inhigh-throughput screening campaigns withtens of thousands of data the throughput requirements, withthe westernblotformat being suitablefor about10–100samplesperday and person, compatibility withavailableaffinity reagents, asoutlined above. A key distinguishing factor betweenthese formats is (option B)format. The decision onwhich format tousedepends onthe experimental setupinthe laboratory and several methods. Herein wedescribe twovariants basedonawesternblot(option A)and onahomogeneous AlphaScreen 17| Detection of soluble protein on ice for a maximum of 1–2 h. transferring the supernatant. ready for analysis withthe detection method of yourchoice. 16| ? disturbing the pellets. Keep the samplesonice inacooling block. debris together withprecipitated and aggregated proteins. Carefully remove the tubesfrom the centrifuge and avoid 15| ? (4 °C)ice afterthe lastthawing step. uniform temperature betweentubes. The tubesare vortexed briefly aftereachthawing. The resulting celllysatesare kept on 14| C 13| (viii) A ell ell lysis (xii) (vii)
(iii) TROU TROU (xi) (ix) Wash the nitrocellulose membrane for 10min withwashbuffer. (vi) (iv)
(ii) ) Western blotformat PAUSE PAUSE (x) Blockthe nitrocellulose membrane withblocking bufferfor 1hatroom temperature orovernight at4°C. (v) CR (i) As already described, the detection and quantification of the remaining solubleprotein canbeachieved using Carefully transfer 90 Briefly vortex the tubesand centrifuge the celllysate–containing tubesat20,000 Freeze-thaw the cellstwice using liquid nitrogen and athermal cyclerorheating blocksetat25°C in order toensure a Snap-freeze the heat-treated cellsuspensions inliquid nitrogen. I fill the chamberwith1× NuPAGE MESSDSbuffer. filled withwashbuffer. all the tubesat70°Cfor 10min. loading bufferinnew 0.2-mlPCRtubes;vortex briefly, briefly spin down the samplesina microcentrifuge and heat T the background ifrequired. manufacturer’s description, and perform the transfer using iBlotgel transfer device incubated withthe primary antibody overnight at 4°C. of 0.4 briefly with deionized water inatray. given temperature. visualize the differences insolubleprotein levelscausedbyincubation withdifferent compounds ateach however, itisconvenient toloadthe samplesfrom the same temperature endpoint next toeachother tobetter the samplesfor eachCETSA melt curveonthe same gel. The loading order of the samplesonthe gel canbevaried; Initiate the SDS-PAGE procedure bymixing 40 Wash the membrane three times for 10minwith wash buffer. Longer and/or additional washcyclescanfurther reduce Briefly rinse the nitrocellulose membrane withwash bufferand incubate them for 1hat room temperature with15ml Remove the nitrocellulose membrane(s) from the transfer sandwiches and placethe membrane inacontainer ortray Initiate the westernblotprocedure byassembling the nitrocellulose iBlottransfer sandwich orstackaccording tothe When the separation of proteins bySDS-PAGE iscomplete, remove the gels from their plastic cassettesand rinse them Perform SDS-PAGE at200Vfor 45minusing anappropriate powersupply. Carefully load13 Assemble precast NuPAGE Novex 4–12%(wt/vol)bis-Tris Midi 26-wellgels inXCell4 SureLock Midi-Cell chambersand Again vortex the tubesand briefly spin down the samplesinamicrocentrifuge. I B B CAL LES LES
PO PO ●
H H I I T STEP µ NT NT IMI OOT OOT g/ml anti-p38 The experiment can be paused here, with the samples kept at −80 °C overnight. Although the samples should be processed on the same day, when they are not in use they should be stored N Be sure not to touch the sides of the tubes and especially not the pellet with the pipette tip when I I N N G 1 h G G µ l (corresponds tothe lysatefrom ~2.6×10 µ
● l of eachsupernatant withthe solubleprotein fraction toanew tube. The solublefraction isnow
α T IgG rabbit primary antibody dilutedinblocking buffer. Alternatively, the membrane canbe IMI N G 5–6h(or1–2dwithovernight incubations) µ l of eachrespective clarifiedcelllysatewith20 4 HL-60cells)of eachsampleinto the wells. Run natureprotocols g for 20minat4°Ctopellet cell ‘ Program 1’(25V, 8min). µ
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© 2014 Nature America, Inc. All rights reserved. (part 1of the PROCEDUREsection). to perform this experiment isderived from analysis of the data obtained ingenerating the apparent melting curve a dose-response fingerprint at50°Conthe basis of 11different compound concentrations. The appropriate temperature the compound concentration isvaried instead of the temperature when heating the cells. Steps 18–30show how toestablish compound treatment P (B) (xviii) 2112 (xvii) (viii) (xiii) (xix) (xvi) (xiv) protocol art art 2, ofdetermination the isothermal dose-response fingerprint for an intracellular protein: cell handling and (vii) (xx) (xv) (iii) (ix) (vi) (iv)
(ii) (v) CR (i) A
| I VOL.9 NO.9VOL.9 lpha for ~5minat500r.p.m. ? suitable model. SeeStep17A(xix)for details. blocking bufferfor 1hatroom temperature. and mixthe contents carefully bysixrepeated aspiration and dispensing steps. T p38 of consecutive images until the bands are overexposed. Forthisparticular setup,the bands corresponding to see the ‘Limitations’ section. compound. Forfurther details onthe choice of temperature and quantitative interpretations of the thermal shifts, majority of the protein remains solubleinthe presence of asaturating concentration of aknown stabilizing a majority of the protein isprecipitated and not detected inthe absence of stabilizing compound, butatwhich a for follow-up isothermal dose-response orscreening experiments. This is commonly done atatemperature atwhich (AlphaScreen donor beads). Inspect the curvetoensure compliance withthe model and withthe intention tochoose anappropriate temperature Use adata processing software (e.g., GraphPad Prism)and evaluate the chemiluminescence data byapplying a Read the chemiluminescence signal inthe Envision platereader. Incubate the plateatroom temperature for atleast 2horpreferably overnight. Add 2 Incubate the platefor 2hatroom temperature. Add 5 Transfer 4 Dilute eachrespective cleared celllysate15-fold in1×PerkinElmer lysisbuffer(from the AlphaScreen SureFire kit) Inspect the curvetoensure compliance withthe model and withthe intention tochoose anappropriate Use adata processing software program (e.g., GraphPad Prism)and evaluate the data byapplying asuitable Choose anappropriate signal accumulation mode setupinImage Lab, expose the membrane and acquire aseries Lift the membrane using tweezers, allowthe excess ECLsolution todrip onto acleanpapertowel,place the Use acleantweezertoliftand placeeachmembrane onacleanflatsurface (such asaplastic lid) and add 3ml of Wash the membrane three times for 10minwithwashbuffer. Longer and/or additional washcyclescanreduce the Incubate the membrane with15mlof 80ng/ml bovine anti-rabbit HRP-conjugatedIgG secondary antibody dilutedin ? within GraphPad Prism. respectively. Datawere then fittedtoobtain apparent RESULTS, data were first normalized bysetting the highest and lowestvalueineachsetto100and 0%, model (seethe ‘Limitations’ section regarding model choice). Forthe melting curvesshown inthe ANTICIPATED MP imager. membrane betweenthe sheets of aplastic film(e.g., clearsheet protectors) and placethese into the ChemiDoc background ifrequired. Agitate the plateonashaker for ~5minat500r.p.m. compound. Forfurther details onthe choice of temperature, seethe ‘Limitations’ section. a majority of the protein remains solubleinthe presence of asaturating concentration of aknown stabilizing which amajority of the protein isprecipitated and not detected inthe absence of stabilizing compound, butatwhich temperature for follow-up isothermal dose-response orscreening experiments. Thisiscommonly atatemperature at solution isevenlycovering the entire membrane. Ifpreferred, develop the membrane separately. Clarity westernECLsubstrate solution toeachmembrane; incubate for 5minatroom temperature. Make sure thatthe volume toolsinthe analysis toolbox inImage Lab. Select animage inwhich none of the bands are overexposed, and then quantify the different p38 I
CAL TROU
TROU CR α S creen format should appearrapidly and become overexposed within5–10s. I The procedure for establishing anITDRF T µ µ I B B l of acceptorbeadmix,sealthe platewithaThermowell platesealand agitatethe plateonashaker l of donor beadmix,sealthe plateand centrifuge the platebriefly at100 CAL LES LES | 2014 µ l of eachsolution from the PCRtubestoaseparate wellof a384-wellProxiPlate.
H H STEP OOT OOT |
● natureprotocols From now on,allworkmust beperformed under subdued light becauseof light-sensitive reagents I I
N T N ● IMI G G
T IMI
N G 2 h N G 5–6horovernight
CETSA
issimilartothe apparent melting curveexperiment, except that T agg valuesusing the Boltzmann Sigmoid g atroom temperature for 10s. 7 equation α bands byusing the
© 2014 Nature America, Inc. All rights reserved. ( i.e., awesternblotapproach (option A)and ahomogeneous AlphaScreen-based assay(option B). 30| C consistent cooling between wells. 29| ? and hence it will not affect the total incubation time substantially. 28| Heat treatment of cell suspensions compound accesstothe cells. 27| 26| 25| 24| the copies ina−20°Cfreezer asbackup. 23| wells. Column12withadded DMSO servesasanegative control. in allwells. Thisprocedure generates an11-point dose-response curvewiththreefold difference inconcentration between until the sampleincolumn11hasbeenthoroughly mixed. Remove 5 compound solutions using amultipipette. Continue thisprocess bymoving one columnatthe time, i.e., 2–3,3–4and soon, predispensed DMSO byaminimum of sevenaspiration and dispensing steps. Thisisdone simultaneously for allthree 22| highly skin-permeable. when and you important are mutagenic handling is DMSO This compounds. particularly solutions, as the is solvent ! of column1of a96UNUNCplate. Place10 21| milliliter. the culture medium. Resuspend the cellpelletinfresh cellculture medium toyield acelldensity of ~40 million cellsper 20| 19| western blot–and AlphaScreen-based detection. procedures and supplies. Approximately 60million HL-60cellsare needed for three ITDRF 18|
A ell ell lysis and detection of soluble protein
CAUT TROU
(ii) ) Western blotformat CR (i) Forreasons already described, wepresent twodifferent means for the detection and quantification of solubleprotein, Remove the platefrom the instrument and place it inanaluminum blockat room temperature for 3mintoensure Placethe PCRplateinaTECHNEthermal cyclerand heat the cellsfor 3minat 50 °C. Incubate the platefor 30mininthe CO Add 15 Transfer 5 Diluteallthe serially dilutedsolutions 50-fold through the addition of cellculture medium. Splitthe serial dilutions into twocopies bytransferring 5 Serially dilutethe stocksolutions bytransferring 5 Place15 Centrifuge the conical tubestopelletthe cellsat300 Collectthe cellsuspension withaserological pipetteand transfer the cellsto15-mlconical tubes. Expand HL-60cellsincellculture medium toacelldensity of ~1–2million cellspermlusing standard sterilecellculture I handling). in strips. Thisyields atotal of 36PCRtubesor12 percompound (keeping the tubesinstripsfacilitates tube T Snap-freeze the heat-treated cellsuspensions inliquid nitrogen. Transfer 50 I I B ON
CAL LES PAUSE Use appropriate safety equipment and Use safety a appropriate environment controlled toxicwhen potentially with working µ
H STEP l of the homogenous cellsuspension (40million cellspermilliliter)toeachwellof the Twin.tec PCRplate. µ OOT µ l of the 4mMDMSO stocksolutions of the AMG-548,SB203580and ERK11ecompounds inseparate wells
PO l of alldilutedcompound solutions toaTwin.tec PCR plate. In this instrument, the plate must be present during temperature ramping, but this is achieved in 5 s I µ N I l of the samplesinindividual wellsof the Twin.tec PCRplateinto individual 0.2-mlPCRtubes, preferably NT G The experiment canbe paused here withthe samples kept at−80°Covernight.
●
T IMI N G 5–6h(or1–2dwithovernight incubations) ●
T IMI µ 2 N l of DMSO incolumns 2–12of the same plate. incubator at37°C.Carefully shake the platemanually every10mintopromote G 10 min µ l from columns 1to2and bymixing thoroughly withthe g for 3minatroom temperature and carefully remove and discard µ l of allsolutions toasecond 96UNUNCplate. Place one of µ
l from column11toensure the same volume of 10 natureprotocols CETSA experiments using both
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µ l © 2014 Nature America, Inc. All rights reserved. in the protocol. Steps 17A(xix),17B(viii),30A(vi) or30B(ix).Specificconsiderations havealso beenadded toaddress specific steps Kn manufacturer’s troubleshooting guide ( troubleshooting guides. Similarly, for concerns related tothe AlphaScreen technology, the userisreferred tothe techniques, respectively. Forgeneral problems related towesternblotting procedures, the userisdirected toavailable performing the CETSA procedure, aswellduring target protein detection bymeans of westernblotand AlphaScreen Troubleshooting advice canbefound in ? (B) 2114 (viii) Readthe luminescence signal inthe Envision platereader. protocol (vii) Incubate the plateatroom temperature for at least2horpreferably overnight.
(iii) (iii) TROU (ix) (iv) (vi) (iv) (vi) The problems listedinthe tableare generally observedwhen the potential stabilization isbeing detected, i.e., in (ii) owledgebase/AlphaScr (v) (v) (i) A
| VOL.9 NO.9VOL.9 lpha ProxiPlate. ? are kept onice (4°C)afterthe lastthawing step. uniform temperature betweentubes. The tubesare vortexed briefly aftereachthawing. The resulting celllysates ? cycles of 25 the 96-wellhead onthe CyBi-Well liquid handling station. Thisisachieved bymeans of sixaspiration and dispensing on streptavidin-coated beads. to minimizeinterference of biotin inthe cellmedium withthe detection step,asone of the antibodies iscaptured checked thatthe medium components do not interfere withthe readout. Inthisparticular case, the dilution serves and the volume of added celllysisbuffer(ongoing efforts inourlaboratory). When attempting this, itmust be for 10s. Agitate the plate(s)onaplateshaker for ~5 minat500r.p.m. Use a multipipette to add 2 ? model. SeeStep30A(vi)for details. plate onashaker for ~5minat500r.p.m. ? the choice of models). We applythisequilibriummodel for empirical ranking of compounds (see‘Limitations’ section for adiscussion on applying the saturation binding curve(rectangular hyperbola; binding isotherm) function withinGraphPad Prism. normalized toamaximum of 100%for the highest valueand to0%for the lowestvalueand then analyzed by to disturbthe pellet.Keep the samplesonice inacooling block. debris together withprecipitated and aggregated proteins. Carefully remove the tubesfrom the centrifuge soasnot Use amultipipette toadd 5 Using the same 96-wellhead, transfer 4 Thoroughly mixthe cellsuspensions withthe added lysisbuffertoachieve acompletecelllysisinallsamplesbyusing Add 140 From here on,the protocol isidentical tothatdescribed inStep17A.However, for analysis, data should be For eachtube, transfer 40 Briefly vortex the tubesand centrifuge the celllysatecontaining tubesat20,000 Freeze-thaw the cellstwice using liquid nitrogen and athermal cyclerorheating blocksetat25°Cto ensure a Use data processing software (e.g., GraphPad Prism)and evaluate the chemiluminescence data byapplying asuitable Incubate the platefor 2hatroom temperature. (AlphaScreen donor beads). transferring the supernatant. fraction isnow ready for analysis bywesternblotting. B
TROU
TROU TROU TROU
LES CR CR CR S creen format I I I H T T T I B I I B B B OOT CAL CAL CAL LES LES µ LES LES | 2014 l of 1×PerkinElmer lysisbuffertoallwells. I
µ H H N H H STEP STEP STEP l each. OOT OOT G OOT OOT | natureprotocols Forcostand throughput reasons, there willbeinterest inminimizing boththe samplevolume From now on,allworkmust beperformed under subdued light becauseof light-sensitive reagents Besure not totouch the sides of the tubesand especially not the pelletwiththe pipettetipwhen I I I I een-SureFire-no-wash
N N N N ● G G
G G
T
IMI µ N µ l of eachsupernatant containing the solubleprotein fraction toanew tube. The soluble G µ l of donor bead mix, seal the plate(s) and centrifuge it briefly at 100 5–6horovernight l of the acceptorbeadmix,sealthe platewithThermowell platesealsand agitatethe http://www.perkinel T able
-assay/surefire_trou 1 µ , withseparate sections for general issuesthatmay arisewhen l of the samplesfrom the Twin.tec PCRplatetoa quadrant of the 384-well
mer.com/se/Resources
bleshoot.xhtm l ). /TechnicalResources/ g for 20minat4°Ctopellet cell ApplicationSupport g at room temperature
-
© 2014 Nature America, Inc. All rights reserved. T a 6 4 Apparent melting curve and ITDRF S 8 tep b le le 1 1 |
Troubleshooting table. Wide samples is observed for treated thermal stabilization No or little samples is observed for treated thermal stabilization No or little cell viability Low cell count, low samples is observed for treated thermal stabilization No or little P roblem T agg range
CETSA Potential multidomain protein Compound does not bind target protein protein Compound does not stabilize the target form detected by the antibody different form of the target than the Compound binds and stabilizes a Co-factors are required for binding equilibrium rearrangements Fast compound off-rate and thus is exceeded The linear range of the detection system Compound concentration is too low Compound is not cell permeable compound High compound concentration, toxic Poor compound solubility and stability Inadequate incubation time P ossible reasons
experiment, experiment, general considerations
and cell toxicity profile tolerates this Increase the compound concentration if the solubility the apparent Try TSA on the cell lysate or purified protein and compare compound exposure for the cells Decrease compound concentration; shorten the time of buffer or medium another solvent or incubating the cells in a modified Check compound stability and solubility. Consider using modifications and activations of compound, if applicable Increase the incubation time to allow metabolic S the precipitation is slow complete precipitation. Try to increase the heating time if retain some of the target protein in solution, preventing individual case. Interactions with other proteins could full-length protein must be carefully examined for each to which each domain results in precipitation of the different thermal stabilities between domains. The extent Inherent melting behavior of multidomain proteins with or the addition of co-factors? Does the compound binding require an activation event Try another compound as a positive control, if available. be stabilized by endogenous ligands TSA assays). Furthermore, the target protein may already not occur (compare with false negatives in traditional There are proteins for which thermal stabilization does Try other compounds as positive controls, if available. that could influence the status of the target protein tion strategies, e.g., MS. Investigate different cell states Screen for alternative antibodies or use alternative detec gated biology Add the relevant co-factors, depending on the investi in cell culture medium or buffer during the heating step Skip washing of cells, i.e., keep the compound present linearly to decreases and increases in concentration centration is within a range in which the signal responds Dilute the samples to ensure that the target protein con outside of the linear range of the detection reagents. peratures or even masked if the detection is performed A thermal transition may be shifted toward higher tem olution T agg natureprotocols with that obtained in intact cells
| VOL.9 NO.9VOL.9 protocol | 2014 (continued)
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- © 2014 Nature America, Inc. All rights reserved. T 2116 a S 30B(ii) 14, Apparent melting curve and ITDRF Apparent melting curve and ITDRF 30A(iii) 14, 11,12 11 protocol tep b le le
| VOL.9 NO.9VOL.9 1 1 |
Troubleshooting table (continued). Low Low signal Hook effect between replicates Uneven signal No transition states between replicates Uneven signal samples is observed for treated thermal stabilization No or little temperature samples at the lowest treated and control between compound- in signal intensity Large differences P roblem | 2014
| natureprotocols CETSA CETSA Insufficient cell lysis antibody of similar molecular size, low affinity ing of target protein by other proteins High cell lysate concentration, mask Uneven cell lysis Wrong Poor compound solubility and stability Liquid handling problems Uneven heating and or cooling Too-large temperature increments degradation of target proteins Altered cellular processes leading to target protein induced by the compound. Upregulation or downregulation of Membrane-associated protein denatured forms of the protein The antibody recognizes several partly P ossible reasons
experiments experiments using AlphaScreen detection experiments using western blot detection T agg range
-
small thermal shifts Decrease the temperature increments to visualize possible purified protein and compare the compound exposure time. Try TSA on the cell lysate or Decrease the compound concentration and/or shorten the to isolate the transition of interest Attempt different lysis buffer compositions and protocols to be affected by the means of membrane anchoring. Thermal denaturation followed by precipitation is likely centrifuge at greater force and for a longer time denatured protein material. Increase heating time, aggregates or to promote precipitation of partly Try different lysis buffers to avoid resolubilizing S facilitate facilitate cell lysis freeze/thawing cycles. Optimize mixing protocols to detergent compositions or consider the addition of Optimize cell lysis procedures, e.g., try different higher affinity toward the target protein as it can mask the epitopes. Try another antibody with ‘hidden’ targets. Lower the nonfat milk concentration, primary antibody to allow time for the antibody to access resolution in the desired area. Incubate longer with the separation. Use SDS-PAGE gels that allow better Run the SDS-PAGE for a longer time, allowing better Dilute the cell lysate before running the SDS-PAGE. mixing protocols to facilitate cell lysis detergent compositions or freeze-thaw cycles. Optimize Optimize cell lysis mixing procedures, e.g., try different increments Investigate lower temperatures; try smaller temperature modified buffer or medium using another solvent or incubating the cells in another Check compound stability and solubility. Consider as expected equipment are properly calibrated and performing Ensure that all pipetting steps and liquid handling both heating and cooling heating and cooling block temperature is uniform during Check the calibration of the instrument. Ensure that the olution T agg with intact cells (continued)
© 2014 Nature America, Inc. All rights reserved. Box 1 Step 30B, AlphaScreen format: 5–6 h or overnight Step 30A, western blot format: 5–6 h (or 1–2 d with overnight incubations) Steps 28 and 29, heat treatment of cell suspensions: 10 min Steps 18–27, cell handling and compound treatment: 2 h Step 17B, AlphaScreen format: 5–6 h or overnight Step 17A, western blot format: 5–6 h (or 1–2 d with overnight incubations) Steps 14–16, cell lysis: 1 h Steps 11–13, heat treatment of cell suspensions: 10 min Steps 1–10, cell handling and compound treatment: 2–3 h ● T Box 4 a S 28 15
tep b T le le IMI , , high-throughput screening procedure: 7–8 h or overnight , antibody and cell line screen: 5–6 h after stable cell lines are acquired (or 1–2 d with overnight incubations) 1 1 N | G
Troubleshooting table (continued). the plate Uneven signal over High background High background temperature samples at the lowest treated and control between compound in signal intensity Large differences P roblem gradients during reading Plate edge effects owing to temperature gradients during compound treatment Plate edge effects owing to temperature the aggregated protein Detergents in the lysis buffer dissolve protein Antibodies also recognize aggregated Cross-reactivity in the detection system quenching of antibody recognition recognition, i.e., a compound-induced Interference of compounds on epitope librium Antibody binding has not reached equi Interference from biotin in the media limit of detection Target molecule concentration below the the linear range of detection Target molecule concentration is above P ossible reasons - attempt attempt the identification of alternative buffer conditions Evaluate alternative antibody pairs or alternatively establishment of equilibrium alternative antibody pairs that allows a faster Incubate the plate for a longer time period. Evaluate concentration Alternatively, increase the AlphaScreen donor bead or increase the dilution of samples to reduce interference. on your detection system. If necessary change medium Check the influence of the cell media composition within the linear range of detection Titrate cell number and make sure that your analyte is (hook effect) detection system and generate a decreased signal too high analyte concentration may oversaturate the is within the linear range of detection. Note that a Titrate cell numbers and make sure that your analyte S See See also TROUBLESHOOTING guidance for Step 6 above equilibration close to the instrument before reading ment ambient temperature or allow a proper time for Ensure that incubation with donor beads is at the instru compound dilution and cell treatment Keep temperature constant in media and reagents during avoid solubilizing aggregates Optimize the lysis buffer composition and protocol to tion or filtration step to remove aggregated proteins Evaluate alternative antibody pairs. Include a centrifuga dilution of samples to reduce interference recognition of the target protein. If possible, increase the Evaluate alternative antibody pairs with more selective See also TROUBLESHOOTING guidance for Step 6 above during detection that prevents ligand binding olution natureprotocols
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- - © 2014 Nature America, Inc. All rights reserved. Fig. 50 °C,i.e., atatemperature atwhich the majority of unliganded protein isdenatured and precipitated (green curvesin define the temperature atwhich topursueITDRF are differently recognized bythe twodetection formats (see‘Limitations’). Onthe basisof these results, the userneeds to ligand-bound orunliganded protein inthe homogeneous format orwhether there are folding intermediates availablethat test systems inwhich thisisnot the case, itisnecessary todefine whether this results from differential recognition of ure the same protein subpopulations inthe solublefraction aftercentrifugation toremove celldebris and aggregates. For We take the consistency betweendata asademonstration thatthe westernblotand AlphaScreen detection methods meas tial response inthe presence of 20 creen) for AMG-548(ref. blot) and 53.7±0.6°C(AlphaScreen) for SB203580(ref. give consistent responses betweenthe detection techniqueswithashiftof the apparent 48 ± 0.8 and 46.7 ± 0.7 °C for the western blot and AlphaScreen approaches, respectively. Two established p38 Figure 3 microplate reader canbeplottedversustemperature togivethe same typeof apparent melting curveasillustrated in Similarly, the relative chemiluminescence valuesmeasured using an AlphaScreen-based detection protocol onacompatible fit the data toasuitable model for the transition (seeStep17A(xix)inthe PROCEDURE), resulting in apparent temperature toyield the apparent melting curve, asshown in antibody. The relative intensities of the bands canbequantified using anappropriate camera and plottedasafunction of protein amounts atthe lowesttemperature, the so-calledhook effect,becauseof restricted target protein accessfor the increases ( will observethe presence of the protein atthe lowertesttemperatures followed byitsdisappearance asthe temperature illustrated in The expected results from melting curveexperiments inthe absence (controls) and presence of stabilizing ligands are A representative data from experiments conducted incelllysatesand intissuesamples(seealso All experiments included inthissection were obtained using intact cells. The userisreferred tothe original work ANT 2118 protocol pparent meltingcurve for intracellular p38 with three vehicle-treated mice, and itshows large differences inthe remaining solubleprotease MetAP-2 levels. internal controls of loaded cellamounts byquantifying housekeeping enzymes inaliquots taken before orafterthe heating step. use appropriate controls internally betweensamples, aswellbetweenexperiments. Control experiments could, for example, include be possibletoaliquot toensure thatthe target protein isevenlydistributedbetweensamples. It is, however, of great importance to be affected, aslevelsof the target protein may varyfrom one celltypetoanother; insuch casesahomogenate of such cellswould lations orinwhich there isapossibilityto fractionate cellsby cell type. Fortissuesamplesinwhich several cell typesexist, data could and ITDRF allow alsoother parallel samplecharacterization alternatives thanCETSA melt curves In contrast, tissuehomogenates and lysatescanbescaleddown involume and would ligand-target associations, which couldresult ininaccurate, misleading conclusions. been collected. Such rapid handling of heating step,which allowsfor experiments tobecarried outinstantly aftertissuehas intact tissuealiquots isthatitcircumvents extensive samplepreparation before the approaches allowfor the design of or processed totissuehomogenates orevenlysatesbefore heat challenge. Boththese to sampleextraction. correlation betweentarget engagement incertaintissuescouldbeindicative of target engagement alsointissuesthatare unavailable indicative of the lackof response from apatient ormodel animal toagivendrug orbeusedinabiomarker manner inwhich a the of therapeutic windows, of target effects(such as engagement of hepatic enzymes orothers). The useof CETSA toinvestigate The usefulapplications from such experiments include studies ontissuedistribution, dosing requirements and the identification The abilitytoperform studies ontarget engagement insamplesfrom animal studies orpatient material isanattractive potential. Box 3 The graph aboveshows results from heating of intact tissuesamplesat75 °C from liversof three TNP-470-treated mice compared In the current format, CETSA performs wellontissueswithhighly homogenic cellpopu Harvested tissues(orbiopsies from organs ortumors) couldeither beusedintact I
3 C | in vivo VOL.9 NO.9VOL.9 ). Here itisimportant toretain asufficient difference inprotein levelsbetween nonstabilized and ligand-stabilized I PATE b . The apparent CETSA Fig. 3 distribution and target engagement of drugs wasdemonstrated inthe original publication | D Tissuesamples Figure RESULTS experiments. | 2014 a , right). Becauseof apotential protein crowding, the usermay alsoobserveanapparent decrease in 3 | for anexample model systembasedonthe protein kinase p38 natureprotocols T 2 agg 9; see values(givenasaverages ±s.d.) for p38 S in vivo upplementary Fig.2 µ M of the known ERK1/2inhibitorERK11e(ref. 30;see in vivo or ex vivo sampleshelps avoid disturbing the a usingtheoriginal studies. One advantage of heating CETSA experiments. Inthe present example, using p38 for their chemical structures). We alsoobservedalackof asubstan 2 8) and 60.6±1.0°C(westernblot)and 59.2±0.9°C(AlphaS Figure 3 CETSA α a protocol inHL-60cellsand the absence of inhibitorswasat (left).Finally, appropriate software canbeusedto
- α . First,onthe westernblots the user T agg MetAP2
Relative band intensity (%) S 1 valueto54.8±0.6°C(western 100 upplementary Fig.3 . Inaddition, itcouldbe 50 0 Box TNP-470 Liver tissue/control Liver tissue/20mg/kgTNP-470 3 75 ). α Contro ° , thiswasdone at C l α inhibitors T agg 1 for ). values.
- -
-
© 2014 Nature America, Inc. All rights reserved. as as a negative control and the established p38 β cells. ( Figure 4 did not result inany stabilization withinthe tested concentration range (see (AlphaScreen) for SB203580and 19±7nM(westernblot)and 35±16nM(AlphaScreen) for AMG-548,whereas ERK11e of p38 software is used to fit the data to a suitable model yielding the concentrations at which half-maximal thermalcorresponding stabilization data for the homogeneous assayusing AlphaScreen-based detection canbefound in relative intensities are plotted asafunction of ligand concentration togenerate the ITDRF the ligand. The increased protein levelsare observedvisually onthe westernblot( at atemperature where amajor portion of the target protein denatures and precipitates unless it isthermally stabilizedby ligand concentration isincreased tolevelswhere protein binding saturates. Thisoccursbecausethe experiment isconducted When performing the ITDRF Isothermal dose-response fingerprint for intracellular p38 recognition of linear epitopesafterisolation of the solublefraction are used. kinase pairERK1/2in detection. Such aligand-induced detection signal quench isillustrated and described indetail for another example protein screen format, becauseinthiscasethe protein remains inanative and therefore potentially ligand-binding form alsoduring results inareduced abilityof the affinity reagents to detect the remaining solubleprotein when applying the homogeneous As already described under ‘Limitations’, the usershould alsobeaware thatthere may beexamples inwhich ligand binding elevated temperatures, asthistranslates into aloss of sensitive response toligands such thatweakbinders canbemissed. at high temperatures where nonstabilized protein levelsare low. However, atthe same time itisimportant toavoid too- samples such thatany effectof the ligand canbemeasured withstatistical certainty, thus favoring doing these experiments outlined in the PROCEDURE. experiments. The solid lines represent the best fits of the data to the Boltzmann sigmoid within the GraphPad Prism (bluesoftware. circle) as All positive datacontrols. were obtainedAll experimentsas were performed at three independent occasions, and data are given as theERK average11e (purple, ± s.e.m.inverted triangle) from asthese a negative control ( toward p38 ( Figure 3 data to the saturation binding curve model within the GraphPad Prism software. All data were obtained as outlined in the PROCEDURE. performed at three independent occasions, and data are given as the average ± s.e.m. from these experiments. The solid lines represent the best fits of the a a a -actin -actin levels) or the SureFire assay ( ,
Relative band intensity (%) b Relative band intensity (%) 100 100 ) Quantification was either based on western blotting ( 50 50 0 0 10 a , α
–4 b | | inHL-60cellsisobserved. Inthese examples, the outcome was0.41±0.12 ) ) Quantification was either made using western blot ( Illustration of the remaining amount of p38 Illustration of the amount of stabilized soluble p38 α
40 10 . Data were obtained in the absence (green triangle) and in the presence of several different compounds, including the dual ERK1/2 inhibitor –3 SB203580 AMG-548
43 10 [Compound]
46 –2 Temperature
49 10 –1
52 S ( ERK 11e DMSO µ upplementary Data 10
55 M) 0 (
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61 1 b ) ) directed toward p38 experiments, the useris expected toobserveanincreased presence of target protein, asthe
64 10 2 ERK 11e SB203580 AMG-548
67 α inhibitors SB203580 (red square) and AMG-548 (blue circle) as positive controls. All experiments were
70 α . Thisisnot aproblem ifwesternblotsorother detection formats thatallowthe accessible for detection as a function of the temperature used for heat treatment of HL-60 cells. ERK 11e SB203580 AMG-548 AMG-548 AMG-548 55 40 S upplementary Fig. 3 a ° ° Drug conc. C C
, left; the right image shows raw western blot data for p38 SB203580 SB203580 α . . Data were obtained in the presence of the dual ERK1/2 inhibitor ERK 11e (green triangle) α β β β DMSO DMSO -Actin -Actin -Actin a accessible for detection in the presence of increasing concentrations of compounds in HL-60 p38 p38 p38 , , left; the right image shows raw western blot data for p38
α α α AMG-548 AMG-548 58 43 ° °
SB203580 C SB203580 C
a DMSO DMSO
AMG-548 AMG-548 61 46 ) and the established p38 SB203580 ° SB203580 ° C C DMSO DMSO
AMG-548 AMG-548 64 49
SB203580 ° SB203580 ° C C DMSO DMSO S
upplementary Data AMG-548 AMG-548 67 52
SB203580 ° SB203580 ° C C Fig. 4 b α DMSO DMSO inhibitors SB203580 (red square) and AMG-548
Relative AlphaScreen signal (%) µ 100 natureprotocols M (westernblot)and 0.26±0.11 50 0 10 b a
–4 Relative AlphaScreen signal (%) , right). Afterquantification, the CETSA 100 50 0 10 α –3 curvein ) or the SureFire assay (
40 10 [Compound] α –2 for the dose-response
43 as well as corresponding SB203580 AMG-548 Figure 4 10
46
–1 | Temperature VOL.9 NO.9VOL.9
49 Figure 4 ( µ 10 M)
52 0 b . Appropriate
55 protocol 10 ERK 11e DMSO (
58 1 ° a C) b | (left).The 2014 ) directed
61 10 2
64 ERK 11e SB203580 AMG-548 |
67
µ 2119
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70
© 2014 Nature America, Inc. All rights reserved. 2120 protocol data were normalized bydividing the observedvalue for eachwellwiththe average valueof the control wellscontaining DMSO only. 17. Use data processing software (e.g., GraphPad Prism)toevaluate the chemiluminescence data byapplying asuitablemodel. Here 16. Readthe chemiluminescence signal inthe Envision platereader. 15. Incubate the plate(s) atroom temperature for atleast2horpreferably overnight. ~5 min at 500 r.p.m. donor bead mix, seal the plate(s) and centrifuge them briefly14. atDepending 100 on the plate numbers, use a multipipette or Multidrop Combi mounted with a small tube dispensing cassette to13. Incubate the platesforadd 2hatroom temperature. 2 of the acceptorbeadmix,sealthe plateswithThermowell platesealsand agitatethe platesonaplateshaker for ~5minat500r.p.m. 12. Depending onthe platenumbers, useamultipipette orMultidrop Combimounted withasmall tubedispensing cassettetoadd 5 for each96-wellplate, withplate2being placed inquadrant 2and soonuntil the fourth plateisplacedinquadrant 4. samples inthe first96-wellplatetoquadrant one onthe 384-wellProxiPlate using the CyBi-Well. Thisprocedure is done sequentially lysis inallsamples. Thisisachieved bymeans of aminimum of sixaspiration and dispensing cyclesof 25 11. Byusing the CyBi-Well, mixthe cellsuspensions withthe compounds of the 96-wellplatesthoroughly toachieve completecell step, asone of the antibodies iscaptured onstreptavidin-coated beads. the readout. Inthis particular case, the dilution servestominimizeinterference of biotin inthe cellmedium withthe detection be of interest (on-going efforts inourlaboratory). When attempting this, ensure thatthe medium components do not interfere with 10. Add 140 C cooling betweenwells. 9. Remove the platesfrom the instrument and placethem inaluminum blocksatroom temperature for 3mintoensure consistent not substantially affectthe totalincubation time. 8. Placethe PCRplatessequentially inaTECHNEthermal cyclerand heat the cellsfor 3minat50°C. Heat treatment of cellsuspensions compound accesstothe cells. 7. Incubate the platesfor 30mininthe CO 6. Add 20 all wellsreceived the same amount of DMSO, the Echo isalsousedtodispense DMSO uptoatotalvolume of 100nlinallwells. control, and 20nlof DMSO towellsE–Hincolumn12,serving asanegative control, toeachof the four 96-wellplates. To ensure that tec PCRplatesincolumns 1–11. Add 40nlof the 10mMDMSO solution of SB203580towells A–D incolumn12,serving asapositive 5. Use the Echo 550acoustic liquid dispenser toplace20nlof the 10mMDMSO stocksolutions ineachwellof four separate Twin. compounds. Thisisparticularly important when youare handling DMSO solutions, asthe solvent ishighly skin-permeable. ! from commercial small molecule library vendors usingradation a liquid handlerand precipitation. such as the TheCyBi-Well, preparation preferably of source equippedThe platessource with is platesa commonly384-well are head.achievedsealed and by storedtransfer under of stockcontrolled solutions4. Prepare atmosphere from compound mother conditions sourceplates platespurchased (low oxygenby placing and 10 relative mM DMSO humidity)densitystock of ~30million cellspermilliliter. solutions to prevent of the compound individual deg library compoundsmedium. Resuspendin the cellpelletinfresh cellculturea medium supplementedLabcyte with10%(vol/vol)serumand antibiotics toyield acell 384 LDV plate. 3. Centrifuge the conical tubestopelletthe cellsat300 2. Collectthe cellsuspension withaserological pipetteand transfer the cellsto15-mlconical tubes. detection plate, including an overhead of 10% to accommodate dead volumes when using the automated liquid handlingcells per equipment. ml using standard sterile cell culture procedures and supplies. Approximately 260 million HL-60 cells are 1.needed Expand forHL-60 cellsone in384-well cell culture medium supplemented with 10% (vol/vol) serum and antibiotics to a cell density of ~1.5–2C million to perform the screen experiment is set at 50 °C on the basis of the previous melting curve of the ligand-free form of intracellular p38 treatment and heating and one 384-well ProxiPlate for the AlphaScreen-based detection. As already described, the temperature at whichnegative controls at 10 liquid handling instrumentation. The procedure below describes the analysis of 352 different compounds along with 16 positive and 16 hit compounds from a high-throughput screening campaign. To achieve this, the assay is additionally adapted for the use of automated one concentration, but the same procedure can also be applied for a dose-response characterization of a large number of identifiedparallel active processing of a larger number of plates simultaneously. In this example, the screen concerns the testing of libraryThe compounds screening at procedure is very similar to that described for the ITDRF stabilize Normalized data were then illustrated asafunction of the position inthe 96-wellmicroplate (wellID)using GraphPad Prism. Box 4
ell lysis anddetectionof solubleprotein ell handlingandcompoundtreatment CAUT
CR CR CR
| VOL.9 NO.9VOL.9 I I I T T T I I I I CAL ON CAL CAL µ Use appropriate safetyequipment and acontrolled environment when working withpotentially toxic and mutagenic |
l of the homogenous cellsuspension (30million cellspermilliliter)tothe Twin.tec PCRplatesusing aMultidrop. STEP
High-throughput screening procedure fortheidentificationof µ STEP STEP l of 1×PerkinElmer lysisbuffertoallwellsusing aMultidrop. | 2014 From now on, all work has to be performed under subdued light because of light-sensitive reagents (AlphaScreen Donor beads). Forcostand throughput reasons, minimizing boththe samplevolume and the volume of added celllysisbufferwill Inthisinstrument, the platemust bepresent during temperature ramping, butthisisachieved in5s, and hence will |
µ natureprotocols M compound concentration on one 384-well ProxiPlate. To achieve this, four 96-well plates are used for cell ●
T IMI ● 2
incubator at37°C.Carefully shake the platesmanually every10mintopromote T N ● IMI G
T 10min IMI N G a 2h N G ● 5–6horovernight g for 3minatroom temperature and carefully remove and discard the culture
T IMI g N at room temperature for 10 s. Agitate the plate(s) on a plate shaker for G CETSA 7–8horovernight experiment. The major difference is that the assay allows the µ l each.Transfer 4
µ
l of the µ
l of
α µ . l - © 2014 Nature America, Inc. All rights reserved. interests: details are available in the CO by R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and D.M.M. R.J., D.M.M., H. Axelsson, H. Almqvist and T.L.; and the manuscript was prepared procedure; western blot and the AlphaScreen experiments were conducted by designed the experiments and the high-throughput formatting of the CETSA D.M.M. conceived the study; R.J., H. Axelsson, H. Almqvist, T.L. and D.M.M. AUT PerkinElmer for great support in the development of the AlphaScreen assays. Biology Consortium Sweden was inaugurated. We are also grateful to C. Zaniol at for a generous donation of instruments and compound libraries when Chemical infrastructure for research in the field of Research Council, which funds Chemical Biology Consortium Sweden, a national H. Almqvist and H. Axelsson acknowledge Karolinska Institutet and the Swedish (Vetenskapsrådet) and the Swedish Cancer Society (Cancerfonden). T.L., Institutet (Distinguished Professor Award), the Swedish Research Council A online of version the pape Note: Any and Information Source Data Supplementary files are available in the cells and other reagents required perwell. to 384-welloreven1,536-wellplates. Besides yielding anincreased throughput, thiswouldhelp toreduce the number of miniaturization of the presented protocol isprobably possible, withtransfer of the firsttreatment and heating experiment resulting initsstabilization canbeclearlyidentified inascreen setting using the homogeneous assay format. Additional effect onp38 called CBK200177and CBK107148,were alsosubjectedtothe ITDRF average plusthree standard deviations of the response for allincluded compounds (controls excluded). These compounds, compounds inwellsE06and G08thatappearasweakstabilizers, although they are outside the hitlimitasdefined bythe for the vastmajority of compounds wasveryclosetothatof DMSO-only controls. At 50 whereas ERK11einposition G06cannot bedistinguished from the rest of the inactive compounds. Importantly, the response SB203580 inwellD02and AMG-548inwellE09canbeclearlyidentified asstabilizing hitsatbothscreen concentrations, of the observedfold increase of the AlphaScreen signal afternormalization onthe basisof DMSO-only controls. Both compounds, i.e., at10and 50 plate wasthen testedinthe homogeneous AlphaScreen-based CETSA assayattwodifferent concentrations of the library SB203580 and AMG-548,respectively. Inourlibrary, the 12thcolumnina96-wellplateisreserved for controls. The screen removed three compounds positioned inwellsG06, D02and E09and replaced these with10mMDMSO solutions of ERK11e, as outlined inthe protocol, the usercanexpect toachieve protocol responds tothe presence of asmall setof testcompounds taken from adiversitylibrary ( To illustrate the feasibilityof applying the homogeneous assayfor screen purposes, wetestedhow the homogeneous assay High-throughput screening procedure for theidentification of stabilizers of intracellular p38 apparent melting curveexperiments illustrated in not influence the ability of the antibody pairinthe AlphaScreen assayto recognize the protein (as also observedinthe of solublestabilizedtarget protein, i.e., ligand binding does demonstrate thatbothprotocols measure the same amounts responses observedwiththe twoapproaches servesto Again the excellent consistency between the pharmacological are alsopresented inthisexample asaverages ±s.d. obtained when probing ERK1/2stabilization). The values were obtained as outlined in to H11), whereas the red dots are derived from the 10 are derived from the 50 increase compared with the DMSO-only controls on each plate. The blue dots Figure 5 com/reprints/index.htm at online available is information permissions and Reprints c k M no H PET OR w I
N le
CONTR | G G FI Scatterplot of the screening data after conversion to fold signal dgm NANC IB ents α UT ( I I
AL D.M.M., D.M.M., R.J., M.I. and P.N. acknowledge Karolinska ONS S upplementary Fig.4 l r I . . µ NTERESTS
R.J., R.J., H. Axelsson, H. Almqvist, M.I., T.L., P.N. and M plate (running from A01, A02 and so on through Box 4
The authors declare competing financial . online version of the pape c µ hemical Biology, as well as Biovitrum M, respectively. Example results are shown in ). Taken together, these data demonstrate thatcompounds binding top38 µ M plate. All data http://www.nature. r . Fig. 3
b ).
Z ′
values 9. 8. 7. 6. 5. 4. 3. 2. 1.
940–948 (2006). 940–948 assay.light-scattering-based high-throughput discovery.drug for strategy general Protoc. Nat. stability. protein promote that interactions ligand detect to fluorimetry studies.structural for proteins of optimization stability high-throughputThermoFluor-based ThermoFluor.using stoichiometry and affinity binding ofmeasurements anhydrase: carbonic of stability (1975). (1971). equilibria. binding coupled with transitions (1990). calorimetry.scanning differential using (2013). assay. shift thermal cellular the using tissues and Senisterra, G.A. Senisterra, Pantoliano,M.W. scanning differential of use The M. Vedadi, & H. Berglund,F.H., Niesen, P.Nordlund, & Dekker,N. G.T., Detitta, B.M., Hallberg, U.B., Ericsson, ThermodynamicTodd,M.J. & F.R.Salemme, J.K., Kranz, D., Matulis, binding.Macromolecular J.A. Schellman, melting acid nucleic thermodynamicsof Statistical D.M. Crothers, interactionsprotein ultratight to strong of Study L.N. Lin, & J.F.Brandts, D. Molina, Martinez 3 CETSA 1
between0.5–0.8.To prepare such atestplate, we Fold signal increase 10 15 20 25 0 5 procedure, confirming their thermal-stabilizing A01
2 B01 Figure , 2212–2221 (2007). 2212–2221 , et al. et et al. et C01 Anal. Biochem. Anal. et al. et Screening for ligands using a generic and generic a using ligands for Screening 5 High-density miniaturized thermal shift assays as a as assays shift thermal miniaturized High-density natureprotocols asascatterplot D01 Monitoring drug target engagement in cells in engagementtarget drug Monitoring µ 10 50 M, there are alsotwoadditional Well position µ µ E01 50 M SB203580 M SB203580 a
µ Biochemistry 357 M CBK200177 J. Biomol. Screen. Biomol. J. F01 10 50 , 289–298 (2006). 289–298 , Box Biochemistry µ µ Biopolymers M AMG-548 M AMG-548 G01 Biopolymers
4
| J. Biomol. Screen. Biomol. J.
44 VOL.9 NO.9VOL.9 ). Inthe format 50 H01 50 , 5258–5266 (2005). 5258–5266 , µ Science µ M/10
M CBK107418 29 14
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341 α , 2147–2160 , 10 50 | Hit limit 2014 and µ µ , 84–87 , M plate M plate
11
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2121
© 2014 Nature America, Inc. All rights reserved. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 2122 protocol
A Mol. Biomol. Spectrosc. Biomol. Mol. A biology.in interactionsmolecular probing for tool a as cryptates earth rare using transfer energy resonance fluorescence resolved status. proteogenomics.unbiased (2013). 2901 cell. the inside screen activity-independentbiophysical format.96-well a proteinsin toolbox. proteomics (2012). GB2490404 no. patent UK assay. shift thermal a using hit. inhibitors. promiscuous sensitivity.drug anticancer of modelling cells. cancer in sensitivity determination. structure and crystallization, proteinstability, protein promote (2007). 20523–20528 kinases. Ser/Thr with inhibitors Bazin, H., Preaudat, M., Trinquet, E. & Mathis, G. Homogeneous timeHomogeneous G. Mathis, & E. Trinquet, M., Preaudat, H., Bazin, R.M. Eglen, R.M Branca, Asial, I. Asial, Nordlund,expressionrecombinantP.&soluble ScreeningofforR.K.Knaust, I. Weibrecht, protein target a to binding ligand determining for P.MethodsNordlund, a hits your make to how screens: Designing M. W.P.Namchuk,Walters, & of detection the for assay detergent-based A B.K.Shoichet, B.Y.& Feng, J. Barretina, M.J. Garnett, M. Vedadi, Fedorov,O.
| VOL.9 NO.9VOL.9 Nat. Rev. Drug Disc. Drug Rev. Nat. Curr. Chem. Genomics Chem. Curr. et al. et et al. et et al. et et al. et et al. et et al. et et al. et et al. et Engineering protein thermostability using a generic a using thermostability protein Engineering Proc. Natl. Acad. Sci. USA Sci. Acad. Natl. Proc. | Chemical screening methods to identify ligands that ligandsidentify to methods screening Chemical 2014 A systematic interaction map of validated kinase validated of map interactionsystematic A The use of AlphaScreen technology in HTS: current HTS: in technology AlphaScreen of use The HiRIEF LC-MS enables deep proteome coverage and coverage proteome deep enables LC-MS HiRIEF The Cancer Cell Line Encyclopedia enables predictive enables Encyclopedia Line Cell Cancer The Proximity ligation assays: a recent addition to the to addition recent a assays: ligation Proximity Systematic identification of genomic markers of drug of markers genomic identification of Systematic Exp. Rev. Proteom. Rev. Exp. |
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