DOCSLIB.ORG

Guanine Nucleotide Binding Proteins, N, and Ni (Transducin/Camp/Receptor Coupling/Transmembrane Signaling) P

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Guanine Nucleotide Binding Proteins, N, and Ni (Transducin/Camp/Receptor Coupling/Transmembrane Signaling) P Proc. Nati. Acad. Sci. USA Vol. 82, pp. 727-731, February 1985 Biochemistry Antisera against a guanine nucleotide binding protein from retina cross-react with the f3 subunit of the adenylyl cyclase-associated guanine nucleotide binding proteins, N, and Ni (transducin/cAMP/receptor coupling/transmembrane signaling) P. GIERSCHIK*, J. CODINAt, C. SIMONS*, L. BIRNBAUMERt, AND A. SPIEGEL* *Metabolic Diseases Branch, National Institute of Arthritis, Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, MD 20205; and tDepartment of Cell Biology, Baylor College of Medicine, Houston, TX 77030 Communicated by J. E. Rall, October 2, 1984 ABSTRACT Antisera were produced in rabbits against a against TD cross-reacts with the a subunit of Ni but not of guanine nucleotide binding protein (N protein), transducin, Ns; and (iii) antisera against TD can be used as specific purified from bovine retina. Antiserum AS/1, which recog- probes for the ,B subunits of Ns and Ni in crude plasma mem- nized all three subunits (a, 3, and y) of the holoprotein, was branes. tested for cross-reactivity with the subunits of the adenylyl cy- clase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]-associated stimulatory (Ns) and inhibitory (N.) N EXPERIMENTAL PROCEDURES proteins purified from human erythrocytes. AS/1 showed strong reactivity against the (3subunits of both Ns and N. but Materials. Bovine blood, cerebral cortex, and retinas were failed to cross-react with either the a or y subunits of Ns and obtained from a local slaughterhouse. Human erythrocytes N.. Seven additional antisera against transducin reacted with were either freshly drawn or obtained from outdated bank the 8 subunits but not with the a or y subunits of Ns and N.. A blood. Materials for gel electrophoresis and immunoblotting single antiserum against transducin reacted with the a subunit were obtained from Bio-Rad with the exception of peroxi- of N1 but not of Ns. Immunostaining of the (3subunits of Ns and dase-conjugated goat anti-rabbit Ig (Kierkegaard & Perry) N. was proportional to the amount of ,( subunit blotted and to and radioiodinated staphylococcal protein A (New England the antiserum concentration. Immunostaining of either human Nuclear). The sources of other materials have been cited (4). erythrocyte or bovine cerebral cortical plasma membrane pro- Protein Purification. Retinas were dissected from freshly teins with AS/1 showed a single band, comigrating with the (3 obtained bovine eyes, and ROS membranes were prepared subunit of transducin; this band was absent in bovine erythro- (7). TD was purified from ROS membranes essentially as de- cyte membranes. Estimation of the amount of (3 subunit by scribed (6) except that both isotonic and hypotonic elutions immunoblotting with AS/1 showed that the ( subunit com- were performed six instead of three times. TD was eluted prises "2% of bovine cerebral cortical plasma membrane pro- with hypotonic buffer containing 40 uM guanylylimidodi- tein, =100-fold more than in human erythrocyte membranes. phosphate. Purification of Ns, Ni, and the Mr 40,000 protein These findings provide immunochemical evidence for similar- (which contains the Mr 35,000 and Mr 5,000 subunits of Ns ities in the , subunits and differences in the a and y subunits and Ni) was as described (8). For some experiments (see Fig. of this family of N proteins. Antisera against transducin react 2) a fraction containing both Ns and Ni (after the third specifically with the (3 subunits of Ns and N. in crude plasma DEAE-Sephacel step and before hydroxylapatite chroma- membranes and, thus, can serve as specific probes for the ,3 tography) was used. subunit. Immunization with TD. Six New Zealand White rabbits (AS/1-AS/6) were injected intradermally with 100 tkg of pu- rified TD per animal in complete Freund's adjuvant, fol- Retinal-rod outer-segment (ROS) disc membranes contain a lowed by an injection of 50 ,g in incomplete Freund's adju- guanine nucleotide binding protein (N protein), also termed vant 2 wk later. Some animals received additional booster transducin (TD) (1), that couples light activation of rhodop- injections subsequently. Six other animals (CW/1-CW/6) sin to increased cGMP phosphodiesterase activity (2). TD were injected in an identical way with purified TD treated shows functional (1, 2) and structural (3, 4) homology to the with 6 M urea and 10% mercaptoethanol before emulsion in stimulatory and inhibitory N proteins (Ns and Ni, respective- either complete or incomplete Freund's adjuvant. Animals ly) associated with adenylyl cyclase [adenylate cyclase; ATP were bled before and at intervals after immunization, and pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. Ns and N1 are heat-inactivated sera were collected. intrinsic membrane proteins, present in low abundance in Membrane Preparations. Human and bovine erythrocyte plasma membranes (5). Purification of substantial amounts membranes were prepared by hypotonic lysis as described of either protein has not been feasible with available meth- (9). Bovine cerebral cortex was homogenized with a Teflon ods, nor have antisera been readily produced (5). TD, in con- pestle in buffer (10 mM Tris HCl, pH 7.5/2 mM MgCI2/0.25 trast, comprises as much as 4% of ROS membrane protein M sucrose) and was centrifuged at low speed to remove un- (6) and is readily purified in aqueous buffers (6). Therefore, broken cells and then at 10,000 x g for 10 min to collect a we produced antisera in rabbits against purified bovine TD crude membrane pellet. This was washed twice with buffer, and tested the cross-reactivity of these antisera against Ns resuspended in buffer, frozen, and stored in liquid nitrogen. and Ni. We now report that: (i) most (eight of nine) antisera Electrophoresis. NaDodSO4/polacrylamide gel electro- against TD cross-react with the , subunits but not with the a phoresis (10) and discontinuous NaDodSO4 urea/polyacry- or y subunits of purified Ns and Nj; (ii) a single antiserum Abbreviations: N proteins, guanine nucleotide binding proteins; N, The publication costs of this article were defrayed in part by page charge stimulatory N associated with adenylyl cyclase; N., inhibitory N as- payment. This article must therefore be hereby marked "advertisement" sociated with adenylyl cyclase; TD, transducin, the retinal N pro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tein; ROS, rod outer segments. 727 Downloaded by guest on October 1, 2021 728 Biochemistry: Gierschik et aLPProc. NatL Acad ScL USA 82 (1985) lamide gradient gel electrophoresis (4) were performed as de- A. Coomassie blue B. Immunoblot scribed. Purified proteins were prepared for electrophoresis 1 2 3 4 5 6 as described (4), and membranes were prepared for electro- phoresis by suspending in sample buffer (10) and boiling for 5 mm. 1 2 345 Blotting and Immunostaining. Proteins were transferred 94-- from gel to nitrocellulose paper with constant current (150 67 94 v mA) for 12 hr in a Bio-Rad transblot apparatus (11). After 43 P transfer, the paper was incubated in 10 mM Tris HCl, pH .nO*. -. Was 7.5/500 mM NaCl with 3% gelatin to block nonspecific pro- tein binding. The paper was then incubated for 4-24 hr at 30 - room temperature in the same buffer containing 1% gelatin 20----- v and various dilutions of rabbit antisera. After being washed, 16.9- 43 the paper was incubated with a second antibody (1 ,ug of 14.4- peroxidase-conjugated goat anti-rabbit IgG per ml) for 2 hr at 8.2_-.? room temperature. After repeated washings, papers were 6.2 stained in 8.3 mM Tris HCl, pH 7.5/415 mM NaCl/20% 2.5_-- (vol/vol) methanol/0.015% H202/0.5 mg of 4-chloro-i-naph- thol per ml for 10 min at room temperature. Alternatively, after incubation with the first antibody, papers were washed Ns Ni 40k TD TD 40kNiNs and then incubated with radioiodinated protein A (about FIG. 1. Discontinuous NaDodSO4 urea/polyacrylamide gradient 100,000 cpm/ml) in 50 ml of Tris-buffered saline containing gel electrophoresis of N,, Ni, the M, 40,000 protein, and TD. Ap- 1% gelatin for 45 min at room temperature. The paper was proximately 10 ,tg of each preparation were precipitated and sub- then washed extensively in Tris-buffered saline containing jected to discontinuous NaDodSO4 urea/polyacrylamide gradient 0.05% Tween 20 and dried. Autoradiography was then per- gel electrophoresis as described (4). The gel was stained with Coo- formed with Kodak XAR film with DuPont Cronex image- massie blue. Standards (Mr shown x 10-3) are Pharmacia low mo- intensifying screens at -70'C. Autoradiograms were used to lecular weight standards (lane 1) and cyanogen bromide fragments of identify the relevant band on nitrocellulose blots, and then myoglobin (lane 2). Protein staining shows a (3 subunit of M, 35,000 these were cut out for assay in a gamma spectrometer to and a y subunit of Mr "5000 in each preparation (lanes 3-6). The quantitate immunoreactive protein. Protein concentration molecular weights of the a subunits are 42,000 for N, (lane 3), 40,000 for Ni (lane 4), and 39,000 for TD (lane 6). The M, 40,000 protein (40 was determined as described (12). k) lacks an a subunit. (B) Immunostaining of proteins in A blotted onto nitrocellulose paper. Proteins were subjected to NaDodSO4/ RESULTS polyacrylamide gel electrophoresis (12.5% gel) and then blotted onto nitrocellulose paper and immunostained. A 1:50 dilution of AS/1 We produced antisera in rabbits against TD purified from was used and incubation with the first antibody was for 24 hr at bovine retina. TD is a heterotrimer consisting of a, /, and y room temperature. AS/1 reacted strongly with all three subunits of subunits (1).
Load more

Recommended publications
  • Patterns of Green Fluorescent Protein Expression in Transgenic Plants
    Color profile: Generic CMYK printer profile Composite Default screen Plant Molecular Biology Reporter 18: 141a–141i, 2000 © 2000 International Society for Plant Molecular Biology. Printed in Canada. Publish by Abstract Patterns of Green Fluorescent Protein Expression in Transgenic Plants BRIAN K. HARPER1 and C. NEAL STEWART JR.2,* 1Novartis Agricultural Biotechnology Research, Inc., 3054 Cornwallis Rd., Research Triangle Park, NC 27709; 2Dept of Biology, University of North Carolina, Greensboro, NC 27402-6174 Abstract. Modified forms of genes encoding green fluorescent protein (GFP) can be mac- roscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP vi- sualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing trans- genic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice. Key words: expression patterns, green fluorescent protein, marker genes Patterns of IntroductionGFP expression Harper and Stewart Since the discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria it has become a frequently used tool in biology.
    [Show full text]
  • Supplementary Materials and Method Immunostaining and Western Blot
    Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}.
    [Show full text]
  • Scholarworks@UNO
    University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses Summer 8-4-2011 Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria Avijit Biswas University of New Orleans, [email protected] Follow this and additional works at: https://scholarworks.uno.edu/td Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Biswas, Avijit, "Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria" (2011). University of New Orleans Theses and Dissertations. 446. https://scholarworks.uno.edu/td/446 This Dissertation-Restricted is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Dissertation-Restricted in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation-Restricted has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Identification and characterization of enzymes involved in biosynthesis of different phycobiliproteins in cyanobacteria A Thesis Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemistry (Biochemistry) By Avijit Biswas B.S.
    [Show full text]
  • Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology
    International Journal of Molecular Sciences Review Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology Jean-Denis Pedelacq 1,* and Stéphanie Cabantous 2,* 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France 2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France * Correspondence: [email protected] (J.-D.P.); [email protected] (S.C.) Received: 15 June 2019; Accepted: 8 July 2019; Published: 15 July 2019 Abstract: Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP,that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology. Keywords: fluorescent protein; superfolder; split-GFP; bipartite; tripartite; folding; PPI 1. Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and expression of green fluorescent protein (GFP) color variants in the host organism have already been the subject of several reviews [1–4] and will not be described here.
    [Show full text]
  • Intrinsic Indicators for Specimen Degradation
    Laboratory Investigation (2013) 93, 242–253 & 2013 USCAP, Inc All rights reserved 0023-6837/13 $32.00 Intrinsic indicators for specimen degradation Jie Li1, Catherine Kil1, Kelly Considine1, Bartosz Smarkucki1, Michael C Stankewich1, Brian Balgley2 and Alexander O Vortmeyer1 Variable degrees of molecular degradation occur in human surgical specimens before clinical examination and severely affect analytical results. We therefore initiated an investigation to identify protein markers for tissue degradation assessment. We exposed 4 cell lines and 64 surgical/autopsy specimens to defined periods of time at room temperature before procurement (experimental cold ischemic time (CIT)-dependent tissue degradation model). Using two-dimen- sional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, we performed comparative proteomic analyses on cells at different CIT exposures and identified proteins with CIT-dependent changes. The results were validated by testing clinical specimens with western blot analysis. We identified 26 proteins that underwent dynamic changes (characterized by continuous quantitative changes, isoelectric changes, and/or proteolytic cleavages) in our degradation model. These changes are strongly associated with the length of CIT. We demonstrate these proteins to represent universal tissue degradation indicators (TDIs) in clinical specimens. We also devised and implemented a unique degradation measure by calculating the quantitative ratio between TDIs’ intact forms and their respective degradation-
    [Show full text]
  • Investigations on the Impact of Toxic Cyanobacteria on Fish : As
    INVESTIGATIONS ON THE IMPACT OF TOXIC CYANOBACTERIA ON FISH - AS EXEMPLIFIED BY THE COREGONIDS IN LAKE AMMERSEE - DISSERTATION Zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften an der Universität Konstanz Fachbereich Biologie Vorgelegt von BERNHARD ERNST Tag der mündlichen Prüfung: 05. Nov. 2008 Referent: Prof. Dr. Daniel Dietrich Referent: Prof. Dr. Karl-Otto Rothhaupt Referent: Prof. Dr. Alexander Bürkle 2 »Erst seit gestern und nur für einen Tag auf diesem Planeten weilend, können wir nur hoffen, einen Blick auf das Wissen zu erhaschen, das wir vermutlich nie erlangen werden« Horace-Bénédict de Saussure (1740-1799) Pionier der modernen Alpenforschung & Wegbereiter des Alpinismus 3 ZUSAMMENFASSUNG Giftige Cyanobakterien beeinträchtigen Organismen verschiedenster Entwicklungsstufen und trophischer Ebenen. Besonders bedroht sind aquatische Organismen, weil sie von Cyanobakterien sehr vielfältig beeinflussbar sind und ihnen zudem oft nur sehr begrenzt ausweichen können. Zu den toxinreichsten Cyanobakterien gehören Arten der Gattung Planktothrix. Hierzu zählt auch die Burgunderblutalge Planktothrix rubescens, eine Cyanobakterienart die über die letzten Jahrzehnte im Besonderen in den Seen der Voralpenregionen zunehmend an Bedeutung gewonnen hat. An einigen dieser Voralpenseen treten seit dem Erstarken von P. rubescens existenzielle, fischereiwirtschaftliche Probleme auf, die wesentlich auf markante Wachstumseinbrüche bei den Coregonenbeständen (Coregonus sp.; i.e. Renken, Felchen, etc.) zurückzuführen sind. So auch
    [Show full text]
  • Immunohistochemistry Stain Offerings
    immunohistochemistry stain offerings TRUSTED PATHOLOGISTS. INVALUABLE ANSWERS.™ MARCHMAY 20172021 www.aruplab.com/ap-ihcaruplab.com/ap-ihc InformationInformation in this brochurein this brochure is current is current as of as May of March 2021. 2017. All content All content is subject is subject to tochange. change. Please contactPlease ARUPcontact ClientARUP Services Client Services at 800-522-2787 at (800) 522-2787 with any with questions any questions or concerns.or concerns. ARUP LABORATORIES As a nonprofit, academic institution of the University of Utah and its Department We believe in of Pathology, ARUP believes in collaborating, sharing and contributing to laboratory science in ways that benefit our clients and their patients. collaborating, Our test menu is one of the broadest in the industry, encompassing more sharing and than 3,000 tests, including highly specialized and esoteric assays. We offer comprehensive testing in the areas of genetics, molecular oncology, pediatrics, contributing pain management, and more. to laboratory ARUP’s clients include many of the nation’s university teaching hospitals and children’s hospitals, as well as multihospital groups, major commercial science in ways laboratories, and group purchasing organizations. We believe that healthcare should be delivered as close to the patient as possible, which is why we support that provide our clients’ efforts to be the principal healthcare provider in the communities they serve by offering highly complex assays and accompanying consultative support. the best value Offering analytics, consulting, and decision support services, ARUP provides for the patient. clients with the utilization management tools necessary to prosper in this time of value-based care.
    [Show full text]
  • Elongation Inhibitors Do Not Prevent the Release Of
    RESEARCH ARTICLE Elongation inhibitors do not prevent the release of puromycylated nascent polypeptide chains from ribosomes Benjamin D Hobson1,2, Linghao Kong1, Erik W Hartwick3, Ruben L Gonzalez3, Peter A Sims1,4,5* 1Department of Systems Biology, Columbia University Irving Medical Center, New York, United States; 2Medical Scientist Training Program, Columbia University Irving Medical Center, New York, United States; 3Department of Chemistry, Columbia University, New York, United States; 4Department of Biochemistry & Molecular Biophysics, Columbia University Irving Medical Center, New York, United States; 5Sulzberger Columbia Genome Center, Columbia University Irving Medical Center, New York, United States Abstract Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti- puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise *For correspondence: subcellular translation sites using anti-puromycin immunostaining may be confounded by release of [email protected] puromycylated nascent polypeptide chains prior to fixation.
    [Show full text]
  • Tubulin Or Not Tubulin: Heading Toward Total Protein Staining As Loading Control in Western Blots Christian Moritz
    Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots Christian Moritz To cite this version: Christian Moritz. Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots. Proteomics, Wiley-VCH Verlag, 2017, 17 (20), 10.1002/pmic.201600189. hal- 01900776 HAL Id: hal-01900776 https://hal.archives-ouvertes.fr/hal-01900776 Submitted on 22 Oct 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. This is the pre-peer reviewed version of the following article: Moritz, C. P. (2017), Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots. Proteomics, 1600189. Accepted Author Manuscript. doi:10.1002/pmic.201600189 which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/pmic.201600189/ab stract. If you have access, the final form is recommended as it differs in some parts from the pre-version. This version of the article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Pre-peer reviewed version of a review article published in PROTEOMICS Tubulin or not tubulin: Heading towards total protein staining as loading control in Western blots.
    [Show full text]
  • Blocking with Immunizing Peptide (Bl) Protocol
    BLOCKING WITH IMMUNIZING PEPTIDE (BL) PROTOCOL Non-specific binding of an antibody to proteins other than the antigen can sometimes occur. This is usually more common with polyclonal antibodies, but can also occur with monoclonals as well. To determine which band or staining is specific, an immunizing peptide blocking experiment can be performed. Before proceeding with the staining protocol, the antibody is neutralized (incubated with an excess of peptide that corresponds to the epitope recognized by the antibody). The antibody that is bound to the blocking peptide is no longer available to bind to the epitope present in the protein on the Western blot or in the cell. The neutralized antibody is then used side-by-side with the antibody alone, and the results are compared. By comparing the staining from the blocked antibody versus the antibody alone, you can see which staining is specific: this staining will be absent from the Western blot or immunostaining performed with the neutralized antibody. Materials and Reagents • Blocking buffer (usually TBST plus either 5% non-fat dry milk or 3% BSA for Western blot, or PBS plus 1% BSA for IHC) • Antibody • Blocking (immunizing) peptide • Two tubes • Two identical samples (e.g. a Western blot with two identical lanes, cut in half; two slides containing the cells of interest; etc) Method 1. Determine the optimal concentration of antibody that consistently gives a positive result in your particular protocol. Using that concentration, determine how much antibody you will need for two experiments. a. For example, an antibody is being used successfully in Western blot at 0.5 µg/ml.
    [Show full text]
  • Phycocyanin-Mediated Apoptosis in AK-5 Tumor Cells Involves Down-Regulation of Bcl-2 and Generation of ROS
    Molecular Cancer Therapeutics 1165 Phycocyanin-mediated apoptosis in AK-5 tumor cells involves down-regulation of Bcl-2 and generation of ROS Bobbili V.V. Pardhasaradhi,1 A. Mubarak Ali,1 suppression of apoptosis, which is critical in tumor cell A. Leela Kumari,1 Pallu Reddanna,2 and death. Several mechanisms have been proposed to account Ashok Khar1 for suppression of apoptosis in response to COX-2 over- 1 expression. One of the mechanisms being PGE2-mediated Centre for Cellular and Molecular Biology, Hyderabad, India up-regulation of Bcl-2, which in turn inhibited apoptosis and 2School of Life Sciences, University of Hyderabad, Hyderabad, India (8). Alternatively, arachidonic acid-stimulated apoptosis and COX-2 expression inhibited apoptosis by increasing the conversion of arachidonic acid to PGE (8). Abstract Several non-steroidal anti-inflammatory drugs and se- lective COX-2 inhibitors have been shown to induce C-phycocyanin, which is a major biliprotein of the blue- apoptosis in colon cancer cell lines and transformed green algae, has been shown to possess cyclooxygenase-2 fibroblasts (8, 9). C-phycocyanin has also been shown to inhibitory activity. We have studied the effect of phycocy- possess selective COX-2 inhibitory property (10). Thus, our anin on a rat histiocytic tumor line. AK-5 cells are induced interest was to study the induction of apoptosis by into apoptotic death program when treated with phycocy- phycocyanin in tumor cells and the mechanisms involved anin, which involves the activation of caspase-3. Phyco- in the apoptotic process. We have used a rat histiocytic cyanin-mediated apoptotic death is induced through the tumor model for these studies (11) because AK-5 cells generation of reactive oxygen radicals.
    [Show full text]
  • Comparative Proteomics Kit II: Western Blot Module
    Bio-Rad Explorer™ Comparative Proteomics Kit II: Western Blot Module Catalog Number 1662800EDU explorer.bio-rad.com Note: Kit contains temperature-sensitive reagents. Open immediately and see individual components for storage temperature. Duplication of any part of this document permitted for classroom use only. Please visit explorer.bio-rad.com to access our selection of language translations for Bio-Rad Explorer kit curricula. For technical service, call your local Bio-Rad office or, in the U.S., call 1-800-4BIORAD (1-800-424-6723) Dear Educator: Tapping nature's tool kit Animal immune systems naturally generate antibodies when foreign invaders are detected and tag them for destruction. The ability of antibodies to act like magic bullets and target viral, bacterial, and allergenic antigens in the body also makes them ideal for hunting antigens in bioscience research and diagnostic tests. Western blotting employs antibodies to pinpoint specific proteins of interest in complex protein mixtures such as cell extracts. Because of its accuracy, western blotting is used as the confirmatory diagnostic test for HIV and mad cow disease, or bovine spongiform encephalopathy. Western blotting is used extensively in research to determine the presence of specific proteins, to quantify their expression levels, and to determine whether they have undergone genetic or posttranslational modifications. This surefire method categorically identifies proteins of interest based on two distinguishing features: molecular mass and antibody binding specificity. This western blotting activity allows your students to take Bio-Rad's protein profiler kit to the next level. Students use western blotting to specifically identify myosin light chain from the hundreds of other proteins that comprise the muscle cell extracts of closely and distantly related species of fish.
    [Show full text]