Europe -Diagenodes.a. /[email protected] /info @diagenode.comNorth America // -Diagenode Inc. /[email protected] /[email protected] PROTOCOL disrupt and homogenize tissues and cultured cells in just one step. Bioruptor Protein extraction from Tissues and Cultured Cells using General remarks before starting high ensure to required Efficient areproteins. of Diagenode’syields cells purification. cultured and protein tissues animal or of homogenization etc.) and spectrometry,disruption mass blotting, Western (PAGE, techniques Protein extraction from tissues and cultured cells is the first step for many biochemical and analytical Introduction for tissue disruptionand homogenization: • • • • • • • • • • o fntoa suis eg te td o poenpoen neatos, vi uig ionic using avoid interactions), protein–protein of detergents study andhighconcentrations ofsalt. the (e.g. studies functional For immunosorbent assay andmass spectrometry. electrophoresis,enzyme-linked2D for concentration SDS of the reducetorecommended is yield It the blotting. maximize Western and electrophoresis to SDS bufferfor used extraction be can the extracts SDS to proteins. soluble added be the might and SDS interest run. of proteins be the to of assays nature the to according adjusted be must etc.) buffer Conditions for protein extraction (e.g. use of fresh or frozen tissue, composition of extraction degradation. possibleprocedureextractionproteinthe during block toMix ProteaseInhibitor use Always Multiplexing capability Temperature controlled Reproducible Gentle processing Efficient No contamination between samples Fast andsimple Protease Inhibitor Mix Inhibitor Protease 0.25% Na-deoxycholate 0.25% 1% NP-40 NP-40 1% 150 mM NaCl NaCl mM 150 50 mM Tris-HCl (pH 7.4) 7.4) (pH Tris-HCl mM 50 It is always recommended to optimize the buffer composition depending on a specific specific a on depending composition buffer the optimize to recommended always is It research project research use RIPA buffer as a starting point for optimization: for point starting a as buffer RIPA use buffer: Extraction SDS 0.1 - 2% (optional) 2% - 0.1 SDS ® Standard &Plus Bioruptor
® uses state-of-the-art ultrasound technology to efficientlytechnologystate-of-the-arttoultrasound uses Bioruptor ® offers unique benefits www.diagenode.com 1 Europe -Diagenodes.a. /[email protected] /info @diagenode.comNorth America // -Diagenode Inc. /[email protected] /[email protected] PROTOCOL 3. Required materials andreagents 1. I. Protein extraction from Tissues Protocol 2. • • • • • • • • • • • buffer. Scale accordingly. Add Protease Inhibitor Mix (200x) to the cold protein extraction buffer: 5 µl per 1 ml of extraction Pre-cool Fill theTPXtubeswithProtein Extraction Beads. » » » » » » » » Single Cycle Valve (Cat.No.B02020004)(required for Bioruptor Water Cooler (Cat.No.B02010003;115VorB02010002;230V) Bioruptor A Choose between optionAorB: Bioruptor This protocol has been validated for up to 50 mg of tissue. Do not use more tissue per sample. sample. per tissue more use not Do tissue. of mg 50 to up for validated been has protocol This Reagents for protein quantification (optional) Buffer for protein extraction from tissue orcell lysis (notsupplied) Protease Inhibitor Mix(Cat.No.C12010011orC12010012) B: Protein Extraction kit(Cat.No.C20000020);pre-filled 15mlTPXtubes TPX ml 15 for ml 2 - 1 and tubes TPX ml 1.5 for µl 300 - 100 Always(Diagenode, respectrecommendedused. the be might sonicationvolumes:C30010009) No.: Cat. tubes TPX ml 15 or C30010010-1000) or C30010010-50 No.: (Cat. microtubes Use Diagenode’s TPX tubes for sonication. Depending on the desired final volume, 1.5 ml TPX The recommended quantity of the beads is 200 - 250 mg for 15 ml TPX tubes, 40 - 50 mg for for mg 50 - 40 tubes, TPX ml 15 for mg 250 - 200 is beads the of quantity protein recommended The until -80°C at stored and nitrogen liquid in snap-frozen degradation. be protein can prevent to tissues collection Dissected tissue of time the Minimize A 15 mlsonication accessories for Bioruptor Tube holderfor 1.5mltubes(Cat.No.B01200011) For larger quantity cut the tissue and proceed to the disruption in separate tubes. When When tubes. separate in disruption the to proceed and tissue the cut quantity larger For 1.5 ml TPX tubes. TPX ml 1.5 extraction Keep extracted proteins at-80°C. experiments). proceeding 20 - 50 mg of tissue 15 ml TPX tubes are recommended with a final volume of of volume final a with recommended are tubes TPX ml 15 tissue of mg 50 - 20 proceeding 1 - 2 ml. Less tissue could be sonicated in 1.5 ml TPX tubes with a final volume of 100 - 300 µl. µl. 300 - 100 of volume final a with tubes TPX ml 1.5 in sonicated be could tissue Less ml. 2 - 1 2 1 : : Protein Extraction Beads(CatNo.C20000021)for tissue disruption( not required for cell lysis) for sonication 1.5 ml (Cat. No. C30010010-50 or C30010010-1000) or 15 ml TPX tubes (Cat. No. C30010009) Bioruptor Note: ® ® Standard (Cat.No.B01010001)orPlusB01020001) instructions as shown in the corresponding manual before starting any sonication skip thisstep! If usingpre-filled tubes(Cat.No.C20000020Protein Extraction kit) please ® to 4°C using the
Bioruptor ® ® Standard &Plus(Cat.No.B01200015) Water Cooler (Diagenode, Cat. No. BioAcc-Cool). ® Plus)
ue (tity olw the follow (strictly tubes www.diagenode.com
2 Europe -Diagenodes.a. /[email protected] /info @diagenode.comNorth America // -Diagenode Inc. /[email protected] /[email protected] PROTOCOL 8. 7. 6. 5. 4. 1. II. Protein extraction from Cultured Cells 10. 2. 9. : time sonication Total Power : 4°C 4°C : Temperature : 30 sec ON/30 sec OFF sec ON/30 sec 30 : cycle Sonication to remove anyremaining insoluble material. Transfer4°C at centrifugeformin samplesand rpm 15 tube 14,000 new supernatant at toa the the Stop Vortex tubes briefly and proceed to sonication by using the recommended the in is volume final range: 100-300µlfor 1.5mlTPXmicrotubes and1-2mlforthe 15mlTPXtubes. that sure Make tubes. TPX the to pieces tissue Add Beads. Add the required volume of a cold extraction buffer to the TPX tubes filled with Protein Extraction Pre-cool Bioruptor in smallaliquotsat-80°C. Store proteins needed. if analysis extracts further the and Takequantification the for aliquot an Scale accordingly. Add Protease Inhibitor Mix (200x) to the ice-cold cell lysis buffer: 5 µl per 1 ml of extraction buffer. Transfer thesupernatant containing soluble proteins to anew tube. disruption. » » » » » » » » » » » » » » Different protein concentration assays exist including: absorbance at 280 nm, Lowry Lowry nm, 280 at absorbance including: exist assays concentration protein Different maximum for buffer extraction with once washed be might Beads Extraction Protein The (fresh format sample the on depending required be might optimization the that note Please filled completely always be should holder tube the sonication, of homogeneity guarantee To For Western blotting, cells might be lysed directly in 1x Laemmli buffer. After sonication, sonication, After buffer. Laemmli 1x in directly lysed cells. be 1x10^6 might per the cells buffer lysis optimize to blotting, appropriate an Western necessary of µl be For 100 may it using but buffer recommend We RIPA using validated been has protocol This Assay, Bradford Assay, Bicinchoninic Assay (BCA) etc.. Many commercial kits for protein protein for kits commercial Many etc.. (BCA) Assay Bicinchoninic Assay, Bradford Assay, dilution. sample the to lead will this but protein total of recovery be should time sonication shortest The amount. tissue and type tissue tissue), frozen or tubes. with centrifuge extract at 14,000 rpm for 15 min. Transfer the supernatant to a new tube and boil boil and tube new a to supernatant the Transfer min. 15 for rpm 14,000 at extract centrifuge project. research specific a on depending composition buffer quantification are also available. Please note that measuring the protein concentration in an an in concentration protein the measuring that note Please available. also are quantification (i.e. tissues fibrous with occur may disruption Incomplete damage. protein prevent to chosen for 3 min. The supernatant can be used in Western blot. Note that protein quantification by by quantification protein that Note blot. Western in used be can supernatant The min. 3 for SDS extract requires that the assay is compatible with the detergent and reducing agent in in agent reducing and detergent the with compatible is assay the that requires extract SDS muscles). common methods is not compatible with Laemmli buffer. Laemmli with compatible not is methods common the solution. solution. the : H position (High) (High) position H : Bioruptor ® ® to 4°C usingtheWater Cooler.
fe ec 5 yls vre smls n cek h sml vsal for visually sample the check and samples vortex cycles, 5 each after 5 - 15 cycles cycles 15 - 5
Bioruptor
® with the following settings: www.diagenode.com 3 Europe -Diagenodes.a. /[email protected] /info @diagenode.comNorth America // -Diagenode Inc. /[email protected] /[email protected] PROTOCOL 6. 5. 4. 3. 8. 7. : 4°C : Temperature : time sonication Total Power : 30 sec ON/30 sec OFF sec ON/30 sec 30 : cycle Sonication tp the Stop Vortex tubes briefly and proceed to sonication by using the Add ice-cold cell lysis buffer andresuspend thepellet. Incubate onice for 10min. For suspensioncells: For monolayer cells: at -80°C. protein extracts Store needed. if analysis further the and quantification the for aliquot Takean to remove anyremaining insoluble material. Transfer4°C at centrifugeformin samplesand rpm 15 tube 14,000 new supernatant at toa the Samples shouldbeinsolution(viscosity shouldbereduced) » » 4°C. at Aspiratemin thesupernatant. Repeat2more times.Proceed10 to thestepfor 4. rpm 1,500 at centrifuge and tube TPX a to transfer PBS, cold in pellet the Centrifuge suspension at 1,500 rpm for 10 min at 4°C and aspirate the supernatant. Resuspend aspirate asmuchsupernatant aspossible. Proceed to step 4. and scraper and 4°C cell at formin rpm 10 1,500 cells at Centrifuge tube. transferTPX tocella suspension the a use rinse, final the For PBS. cold with times 3 cells monolayer the Rinse » » » » » » requires that the assay is compatible with the detergent and reducing agent in the solution. solution. the in agent reducing and detergent the with compatible is assay the that requires Different protein concentration assays exist including: absorbance at 280 nm, Lowry Assay, Assay, Lowry nm, 280 at absorbance including: exist assays concentration protein Different density, (cell format sample on depending required be might optimization the that note Please filled completely always be should holder tube the sonication, of homogeneity guarantee To step this at appear may viscosity The Bradford Assay, Bicinchoninic Assay (BCA) etc. Many commercial kits for protein quantification quantification protein for kits commercial Many etc. (BCA) Assay Bicinchoninic Assay, Bradford damage. protein prevent to chosen be should time sonication shortest The etc.). type cell tubes. with are also available. also are : H position (High) (High) position H : Bioruptor
®
after 5 cycles, briefly vortex samples and visually check the samples: check visually and samples vortex briefly cycles, 5 after 5-10 cycles 5-10
Please note that measuring the protein concentration in an SDS extract extract SDS an in concentration protein the measuring that note Please
tl peet n h sml wtot h ProteinExtraction Beads(right). the without sample the in present still is tissue non-disrupted whilecycles 5 after (left) containingDiagenode’s Protein Extraction Beads sample the in observed is disruption Complete for efficient tissue disruption using the using disruption tissue Bioruptor efficient for arerequiredBeads ProteinExtraction Figure1. ® Bioruptor Plus ® with the following settings: www.diagenode.com 4 Europe -Diagenodes.a. /[email protected] /info @diagenode.comNorth America // -Diagenode Inc. /[email protected] /[email protected] PROTOCOL Figure 2.Total proteins effectively extracted from tissues usingBioruptor cell an extract from HeLa in Wholecells is 1;43(3):335-45). loaded Feb muscleas positive 1996 control. Res. HeLa in Neuroscicells were expressedJ Brown,lysed using R. the Bioruptor is I. and HSP90 Quraishi (H. level that lowextremely Note muscle. skeletal and brain liver, in panel), (right respectively, HSP90 and panel) (left GAPDH for observed are kD 90 and kD 37 of bands Expected Figure 3.Western blot analysis ofGAPDH andHSP90proteins intissue andcultured cell extracts. proteins were separated bySDS-PAGE andstained withCoomassie Bluedye. TotalSDS. 2% without or with supplemented bufferRIPA in disrupted were tissues mouse Various Liver + SDS - SDS Brain + SDS - SDS ® Muscle Plus + SDS - SDS www.diagenode.com ® .
5 PRO-PROTEIN-EXTRACT_06_08_13