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Review

Western Blotting (immunoblotting): History, Theory, Uses, Protocol and Problems

ORCID Icon, & ORCID Icon
Pages 99-114 | Received 05 Mar 2022, Accepted 20 Feb 2023, Published online: 27 Mar 2023

References

  • Meftahi GH , BahariZ, ZareiMahmoudabadi A, ImanM, JangraviZ. Applications of western blot technique: from bench to bedside. Biochem. Mol. Biol. Educ.49(4), 509–517 (2021).
  • Mishra M , TiwariS, GomesAV. Protein purification and analysis: next generation western blotting techniques. Expert Rev. Proteomics14(11), 1037–1053 (2017).
  • Moritz CP . 40 years western blotting: a scientific birthday toast. J. Proteomics212, 103575 (2020).
  • Barber KW , RinehartJ. The ABCs of PTMs. Nat. Chem. Biol.14(3), 188–192 (2018).
  • Santos AL , LindnerAB. Protein posttranslational modifications: roles in aging and age-related disease. Oxid. Med. Cell Longev.2017, 5716409 (2017).
  • Ghosh R , GildaJE, GomesAV. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev. Proteomics11(5), 549–560 (2014).
  • Baker M . Antibody anarchy: a call to order. Nature527(7579), 545–551 (2015).
  • Prassas I , DiamandisEP. Translational researchers beware! Unreliable commercial immunoassays (ELISAs) can jeopardize your research. Clin. Chem. Lab. Med.52(6), 765–766 (2014).
  • Taylor SC , BerkelmanT, YadavG, HammondM. A defined methodology for reliable quantification of western blot data. Mol. Biotechnol.55(3), 217–226 (2013).
  • Biscombe CJC . The discovery of electrokinetic phenomena: setting the record straight. Angew. Chemie Int. Ed.56(29), 8338–8340 (2017).
  • Majumder PP . Human genome diversity and its value. In: International Encyclopedia of the Social & Behavioral Sciences.SmelserNJ, BaltesPB (Eds). Pergamon, Oxford, UK, 6991–6995 (2001).
  • Renart J , ReiserJ, StarkGR. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. Proc. Natl Acad. Sci. USA76(7), 3116–3120 (1979).
  • Alwine JC , KempDJ, StarkGR. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl Acad. Sci. USA74(12), 5350–5354 (1977).
  • Towbin H , StaehelinT, GordonJ. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA76(9), 4350–4354 (1979).
  • Burnette WN . “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem.112(2), 195–203 (1981).
  • Burnette WN . Western blotting: remembrance of things past. In: Western Blotting: Methods and Protocols.KurienBT, ScofieldRH (Eds). Springer, NY, USA, 9–12 (2015).
  • Hughes AJ , SpelkeDP, XuZ, KangCC, SchafferDV, HerrAE. Single-cell western blotting. Nat. Methods11(7), 749–755 (2014).
  • Nguyen U , SquagliaN, BogeA, FungPA. The Simple Western™: a gel-free, blot-free, hands-free western blotting reinvention. Nat. Methods8, v–vi (2011).
  • Kang CC , YamauchiKA, VlassakisJ, SinkalaE, DuncombeTA, HerrAE. Single cell-resolution western blotting. Nat. Protoc.11(8), 1508–1530 (2016).
  • Taylor SC , PoschA. The design of a quantitative western blot experiment. BioMed. Res. Int.2014, 361590 (2014).
  • Shehadul Islam M , AryasomayajulaA, SelvaganapathyPR. A review on macroscale and microscale cell lysis methods. Micromachines (Basel)8(3), 83 (2017).
  • MacPhee DJ . Methodological considerations for improving western blot analysis. J. Pharm. Toxicol. Methods61(2), 171–177 (2010).
  • Ngoka LC . Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers. Proteome Sci.6, 30 (2008).
  • Grabski AC . Advances in preparation of biological extracts for protein purification. Methods Enzymol.463, 285–303 (2009).
  • Miskiewicz EI , MacPheeDJ. Lysis buffer choices are key considerations to ensure effective sample solubilization for protein electrophoresis. Methods Mol. Biol.1855, 61–72 (2019).
  • Inoue T , PecenP, MaddalaRet al. Characterization of cytoskeleton-enriched protein fraction of the trabecular meshwork and ciliary muscle cells. Invest. Ophthalmol. Vis. Sci.51(12), 6461–6471 (2010).
  • Bass JJ , WilkinsonDJ, RankinDet al. An overview of technical considerations for western blotting applications to physiological research. Scand. J. Med. Sci. Sports27(1), 4–25 (2017).
  • Murphy RM , LambGD. Important considerations for protein analyses using antibody based techniques: down-sizing western blotting up-sizes outcomes. J. Physiol.591(23), 5823–5831 (2013).
  • Murphy RM , MollicaJP, BeardNA, KnollmannBC, LambGD. Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice. Am. J. Physiol. Heart Circ. Physiol.300(2), H595–H604 (2011).
  • Janes KA . An analysis of critical factors for quantitative immunoblotting. Sci. Signal8(371), rs2 (2015).
  • Schröder S , BrandmüllerA, DengX, AhmedA, WätzigH. Improving precision in gel electrophoresis by stepwisely decreasing variance components. J. Pharm. Biomed. Anal.50(3), 320–327 (2009).
  • Chrambach A , RodbardD. Polyacrylamide gel electrophoresis. Science172(3982), 440–451 (1971).
  • Fritsch RJ , KrauseI. Electrophoresis. In: Encyclopedia of Food Sciences and Nutrition (2nd Edition).CaballeroB(Ed.). Academic Press, Oxford, UK, 2055–2062 (2003).
  • Pincus MR . The physiological structure and function of proteins. In: Cell Physiology Source Book.SperelakisN(Ed.). Academic Press, MA, USA, 18–35 (1995).
  • Al-Tubuly AA . SDS-PAGE and western blotting. In: Diagnostic and Therapeutic Antibodies.GeorgeAJT, UrchCE (Eds). Humana Press, NJ, USA, 391–405 (2000).
  • Smith BJ . SDS polyacrylamide gel electrophoresis of proteins. In: Proteins.WalkerJM(Ed.). Humana Press, NJ, USA, 41–55 (1984).
  • Cubillos-Rojas M , Amair-PinedoF, TatoI, BartronsR, VenturaF, RosaJL. Tris-acetate polyacrylamide gradient gels for the simultaneous electrophoretic analysis of proteins of very high and low molecular mass. Methods Mol. Biol.1855, 269–277 (2019).
  • Haider SR , ReidHJ, SharpBL. Tricine-SDS-PAGE. Methods Mol. Biol.1855, 151–160 (2019).
  • Adamson NJ , ReynoldsEC. Rules relating electrophoretic mobility, charge and molecular size of peptides and proteins. J. Chromatogr. B. Biomed. Sci. Appl.699(1–2), 133–147 (1997).
  • Chiou S-H , WuS-H. Evaluation of commonly used electrophoretic methods for the analysis of proteins and peptides and their application to biotechnology. Analytica Chimica Acta383(1), 47–60 (1999).
  • Kašička V . Electrophoresis proteins. In: Encyclopedia of Analytical Science (2nd Edition).WorsfoldP, TownshendA, PooleC (Eds). Elsevier, Oxford, UK, 460–471 (2005).
  • Garfin DE . One-dimensional gel electrophoresis. Methods Enzymol.463, 497–513 (2009).
  • Kurien BT , ScofieldRH. Western blotting: an introduction. Methods Mol. Biol.1312, 17–30 (2015).
  • Másson M , LauritzenE, HolmA. Chemical activation of nitrocellulose membranes for peptide antigen-antibody binding studies: direct substitution of the nitrate group with diaminoalkane. Electrophoresis14(1), 860–865 (1993).
  • LeGendre N . Immobilon-P transfer membrane: applications and utility in protein biochemical analysis. BioTechniques9(Suppl. 6), 788–805 (1990).
  • Berger ST , AhmedS, MuntelJet al. MStern blotting-high throughput polyvinylidene fluoride (PVDF) membrane-based proteomic sample preparation for 96-well plates. Mol. Cell Proteomics14(10), 2814–2823 (2015).
  • Mahmood T , YangP-C. Western blot: technique, theory, and trouble shooting. N. Am. J. Med. Sci.4(9), 429–434 (2012).
  • Anderson GJ , MCC, KennedyRT. Western blotting using capillary electrophoresis. Anal. Chem.83(4), 1350–1355 (2011).
  • Olsen I , WikerHG. Diffusion blotting: a rapid and simple method for production of multiple blots from a single gel. Methods Mol. Biol.1312, 73–76 (2015).
  • Jensen EC . The basics of western blotting. Anat. Rec.295(3), 369–371 (2012).
  • Kurien BT , ScofieldRH. Other notable protein blotting methods: a brief review. Methods Mol. Biol.1312, 487–503 (2015).
  • Bjerrum OJ , Schafer-NielsenC. Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In: Electrophoresis' 86.DunnMJ(Ed.). VCH, Weinheim, Germany, 315–327 (1986).
  • Lissilour S , GodinotC. Influence of SDS and methanol on protein electrotransfer to immobilon P membranes in semidry blot systems. BioTechniques9(4), 397–398; 400–391 (1990).
  • Heda GD , ShresthaL, ThapaS, GhimireS, RautD. Optimization of western blotting for the detection of proteins of different molecular weight. BioTechniques68(6), 318–324 (2020).
  • Van Oss CJ , GoodRJ, ChaudhuryMK. Mechanism of DNA (Southern) and protein (western) blotting on cellulose nitrate and other membranes. J. Chromatogr.391(1), 53–65 (1987).
  • Pillai-Kastoori L , HeatonS, ShiflettSD, RobertsAC, SolacheA, Schutz-GeschwenderAR. Antibody validation for western blot: by the user, for the user. J. Biol. Chem.295(4), 926–939 (2020).
  • Porstmann B , PorstmannT, NugelE, EversU. Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: horseradish peroxidase, alkaline phosphatase or β-galactosidase?J. Immunol. Methods79(1), 27–37 (1985).
  • Mishra M , TiwariS, GunaseelanA, LiD, HammockBD, GomesAV. Improving the sensitivity of traditional western blotting via streptavidin containing poly-horseradish peroxidase (PolyHRP). Electrophoresis40(12–13), 1731–1739 (2019).
  • Krajewski S , ZapataJM, ReedJC. Detection of multiple antigens on western blots. Anal. Biochem.236(2), 221–228 (1996).
  • West S , SchröderJ, KunzW. A multiple-staining procedure for the detection of different DNA fragments on a single blot. Anal. Biochem.190(2), 254–258 (1990).
  • Krajewski S , TsukamotoMM, HuangX, KrajewskiSB. Nonstripping “rainbow” and multiple antigen detection (MAD) western blotting. In: Detection of Blotted Proteins: Methods and Protocols.KurienBT, ScofieldRH (Eds). Springer New York, NY, USA, 287–301 (2015).
  • Negri D , SestiliP, BorghiM, CiccolellaM, BracciL. Enzyme-linked immunospot assay to monitor antigen-specific cellular immune responses in mouse tumor models. In: Methods in Enzymology.GalluzziL, RudqvistN-P (Eds). Academic Press, 457–477 (2020).
  • Luck RF , ShepardBM, KenmorePE. Evaluation of biological control with experimental methods. In: Handbook of Biological Control.BellowsTS, FisherTW (Eds). Academic Press, CA, USA, 225–242 (1999).
  • Rashid MB , Santiago-VázquezLZ, HongSB. Methods Biotechnol.John Wiley & Sons, Hoboken, NJ, USA (2016).
  • Bio-Rad Laboratories, Inc. . Handcasting polyacrylamide gels. In: Bulletin 6201.Bio-Rad, CA, USA (2014).
  • Sander H , WallaceS, PlouseR, TiwariS, GomesAV. Ponceau S waste: ponceau S staining for total protein normalization. Anal. Biochem.575, 44–53 (2019).
  • Salinovich O , MontelaroRC. Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Anal. Biochem.156(2), 341–347 (1986).
  • Gilda JE , GomesAV. Stain-Free total protein staining is a superior loading control to β-actin for western blots. Anal. Biochem.440(2), 186–188 (2013).
  • Brooks HL , LindseyML. Guidelines for authors and reviewers on antibody use in physiology studies. Am. J. Physiol. Heart Circ. Physiol.314(4), H724–H732 (2018).
  • Fosang AJ , ColbranRJ. Transparency is the key to quality. J. Biol. Chem.290(50), 29692–29694 (2015).
  • Cell Signaling Technology Inc . Tech tips video: milk or BSA? Choosing a blocking protein for western blot (2018). https://blog.cellsignal.com/tech-tips-video-milk-or-bsa-choosing-a-blocking-protein-for-western-blot
  • Hoffman WL , JumpAA. Inhibition of the streptavidin-biotin interaction by milk. Anal. Biochem.181(2), 318–320 (1989).
  • Hartwell RC , NelsonMS, KislanMMet al. An improved western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins. J. Virol. Methods125(2), 187–193 (2005).
  • Lipman NS , JacksonLR, TrudelLJ, Weis-GarciaF. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J.46(3), 258–268 (2005).
  • Heidebrecht F , HeidebrechtA, SchulzI, BehrensSE, BaderA. Improved semiquantitative western blot technique with increased quantification range. J. Immunol. Methods345(1), 40–48 (2009).
  • Pillai-Kastoori L , Schutz-GeschwenderAR, HarfordJA. A systematic approach to quantitative western blot analysis. Anal. Biochem.593, 113608 (2020).
  • Polyansky AA , ZagrovicB. Protein electrostatic properties predefining the level of surface hydrophobicity change upon phosphorylation. J. Phys. Chem. Lett.3(8), 973–976 (2012).
  • Nairn AC , DetreJA, CasnellieJE, GreengardP. Serum antibodies that distinguish between the phospho- and dephospho-forms of a phosphoprotein. Nature299(5885), 734–736 (1982).
  • Gingras AC , GygiSP, RaughtBet al. Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. Genes Dev.13(11), 1422–1437 (1999).
  • Magdeldin S , EnanyS, YoshidaYet al. Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis. Clin. Proteomics11(1), 16 (2014).
  • Zong C , YoungGW, WangYet al. Two-dimensional electrophoresis-based characterization of post-translational modifications of mammalian 20S proteasome complexes. Proteomics8(23–24), 5025–5037 (2008).
  • McDonough AA , VeirasLC, MinasJN, RalphDL. Considerations when quantitating protein abundance by immunoblot. Am. J. Physiol. Cell. Physiol.308(6), C426–C433 (2015).
  • Senichkin VV , ProkhorovaEA, ZhivotovskyB, KopeinaGS. Simple and efficient protocol for subcellular fractionation of normal and apoptotic cells. Cells10(4), 852 (2021).
  • Takatalo MS , KouvonenP, CorthalsG, NymanTA, RonnholmRH. Identification of new Golgi complex specific proteins by direct organelle proteomic analysis. Proteomics6(12), 3502–3508 (2006).
  • Gilchrist A , AuCE, HidingJet al. Quantitative proteomics analysis of the secretory pathway. Cell127(6), 1265–1281 (2006).
  • Gomes AV , ZongC, EdmondsonRDet al. Mapping the murine cardiac 26S proteasome complexes. Circ. Res.99(4), 362–371 (2006).
  • Williamson CD , WongDS, BozidisP, ZhangA, Colberg-PoleyAM. Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane and detergent resistant membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts. Curr. Protoc. Cell. Biol.68, 32721–232733 (2015).
  • Bron R , BunnettNW. Antibodies: friend or foe?Am. J. Physiol. Gastrointest. Liver Physiol.309(9), G717–G718 (2015).
  • Bradbury A , PlückthunA. Reproducibility: standardize antibodies used in research. Nature518(7537), 27–29 (2015).
  • Delpire E . Research antibodies: do not use them to stain your reputation. Am. J. Physiol. Cell Physiol.309(11), C707–C708 (2015).
  • Weller MG . Ten basic rules of antibody validation. Anal. Chem. Insights13, 10.177390118757462 (2018).
  • Yang M , YanJ, WuAet al. Alterations of housekeeping proteins in human aged and diseased hearts. Pflügers Arch.473(3), 351–362 (2021).
  • Bettencourt JW , McLauryAR, LimbergAKet al. Total protein staining is superior to classical or tissue-specific protein staining for standardization of protein biomarkers in heterogeneous tissue samples. Gene Rep.19, 100641 (2020).
  • Kirshner ZZ , GibbsRB. Use of the REVERT(®) total protein stain as a loading control demonstrates significant benefits over the use of housekeeping proteins when analyzing brain homogenates by western blot: an analysis of samples representing different gonadal hormone states. Mol. Cell. Endocrinol.473, 156–165 (2018).
  • Moritz CP . Tubulin or not tubulin: heading toward total protein staining as loading control in western blots. Proteomics17(20), 1600189 (2017).
  • Bordeaux J , WelshA, AgarwalSet al. Antibody validation. BioTechniques48(3), 197–209 (2010).