Аннотация
Вестерн-блоттинг (Вестерн-блот, иммуноблот) представляет собой высокоспецифичный способ идентификации и количественного анализа белков, основанный на их электрофоретическом разделении и детекции с помощью антител. С момента разработки в 1970-х годах метод изменялся и совершенствовался, включая автоматизацию, применение микрофлюидных систем и капиллярных платформ. Существуют различные модификации метода, такие как хемилюминесцентный, флуоресцентный, радиоизотопный и количественный вестерн-блоттинг, а также специальные подходы для анализа взаимодействий белков и белок-нуклеиновых комплексов. Метод широко используется в фундаментальных исследованиях для изучения белков: их экспрессии, детекции изоформ, посттрансляционных модификаций, белковых взаимодействий и субклеточной локализации. В клинической практике он применяется в молекулярной диагностике, онкологии, неврологии, эндокринологии, кардиологии и иммунологии. Несмотря на наличие других, более высокотехнологичных методов, таких как масс-спектрометрия, вестерн-блоттинг сохраняет актуальность благодаря своей универсальности и высокой чувствительности. Будущее метода связано с интеграцией с искусственным интеллектом, улучшением детекции и расширением in situ подходов, что обеспечит его адаптацию к современным требованиям биомедицинской науки. В данной работе представлена краткая история возникновения вестрен-блоттинга, информация о различных модификациях данного метода, его преимущества и недостатки, применение данного метода в научных и клинических исследованиях, а также перспективы развития. Для написания обзора были использованы научные публикации, найденные с помощью трёх крупных информационно-поисковых систем: Scopus (мультидисциплинарная база данных от Elsevier), Web of Science (мультидисциплинарная база данных от Clarivate Analytics) и PubMed (специализированная база данных по медицине и биологическим наукам Национальной медицинской библиотеки США).
Annotation
Western blotting (Western blot, immunoblot) is a highly specific method for identifying and quantifying proteins based on their electrophoretic separation and detection using antibodies. Since its development in the 1970s, the method has been modified and improved, including automation, the use of microfluidic systems and capillary platforms. There are various modifications of the method, such as chemiluminescent, fluorescent, radioisotope and quantitative Western blotting, as well as special approaches for analyzing the interactions of proteins and protein-nucleic acid complexes. The method is widely used in fundamental research to study proteins: their expression, detection of isoforms, post-translational modifications, protein interactions and subcellular localization. In clinical practice, it is used in molecular diagnostics, oncology, neurology, endocrinology, cardiology and immunology. Despite the availability of other high-tech methods, such as mass spectrometry, Western blotting remains relevant due to its versatility and high sensitivity. The future of the method is associated with integration with artificial intelligence, improved detection and expansion of in situ approaches, which will ensure its adaptation to the modern requirements of biomedical science. This paper presents a brief history of the emergence of Western blotting, information on various modifications of this method, its advantages and disadvantages, the use of this method in scientific and clinical research, as well as development prospects. To write the review, we used scientific publications found using three major information retrieval systems: Scopus (a multidisciplinary database from Elsevier), Web of Science (a multidisciplinary database from Clarivate Analytics), and PubMed (a specialized database on medicine and biological sciences of the US National Library of Medicine).
Key words: review; Western blotting; antibody; protein separation
Список литературы
ЛИТЕРАТУРА/REFERENCES
Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U S A. 1979; 76(9):4350-4. DOI: 10.1073/pnas.76.9.4350.
Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0.
Switzer R.C. 3rd, Merril C.R., Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal. Biochem. 1979; 98(1):231-7. DOI: 10.1016/0003-2697(79)90732-2.
Southern E.M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 1975; 98(3):503-17. DOI: 10.1016/s0022-2836(75)80083-0.
Alwine J.C., Kemp D.J., Stark G.R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5350-4, DOI: 10.1073/pnas.74.12.5350.
O’Farrell P.H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 1975; 250(10):4007-21.
Moritz C.P. 40 years Western blotting: A scientific birthday toast. J. Proteomics. 2020; 212:103575. DOI: 10.1016/j.jprot.2019.103575.
Sule R., Rivera G., Gomes A.V. Western blotting (immunoblotting): history, theory, uses, protocol and problems. Biotechniques. 2023; 75(3):99-114. DOI: 10.2144/btn-2022-0034.
Krisnawati D.I., Liu Y.C., Lee Y.J., Wang Y.T., Chen C.L., Tseng P.C. et al. Functional neutralization of anti-IFN-γ autoantibody in patients with nontuberculous mycobacteria infection. Sci. Rep. 2019; 9(1):5682. DOI: 10.1038/s41598-019-41952-1.
Tsuji Y. Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin). PLoS One. 2020 Jul 9; 15(7):e0235563. DOI: 10.1371/journal.pone.0235563.
Xu D., Mane S., Sosic Z. Characterization of a biopharmaceutical protein and evaluation of its purification process using automated capillary Western blot. Electrophoresis. 2015; 36(2):363-70. DOI: 10.1002/elps.201400380.
Hagyousif A., Voon Y., Hiroki C. Development of a novel protein multi-blotting device. J. Biomed. Sci. Eng. 2010; 03(12):1125-32. DOI: 10.4236/jbise.2010.312146 .
Degasperi A., Birtwistle M.R., Volinsky N., Rauch J., Kolch W., Kholodenko B.N. et al. Evaluating strategies to normalise biological replicates of Western blot data. PLoS One. 2014 Jan 27; 9(1):e87293. DOI: 10.1371/journal.pone.0087293.
Pillai-Kastoori L., Schutz-Geschwender A.R., Harford J.A. A systematic approach to quantitative Western blot analysis. Anal. Biochem. 2020; 593:113608. DOI: 10.1016/j.ab.2020.113608.
DeNies M.S., Liu A.P., Schnell S. Seeing beyond the blot: A critical look at assumptions and raw data interpretation in Western blotting. Biomol. Concepts. 2024; 15(1):10.1515/bmc-2022-0047. DOI: 10.1515/bmc-2022-0047.
Liu P., Na N., Liu T., Huang L., He D., Hua W. et al. Ultrasensitive detection of ferritin in human serum by Western blotting based on quantum dots-labeled avidin-biotin system. Proteomics. 2011; 11(17):3510-7. DOI: 10.1002/pmic.201000742.
Wang Y., Li Z., Yu H. Aptamer-based Western blot for selective Protein recognition. Front. Chem. 2020; 8:570528. DOI: 10.3389/fchem.2020.570528.
Xu L., Xie H., Wang B., Zhu Z., JiangH., Duang X. et al. Multiplex protein profiling by low-signal-loss single-cell Western blotting with fluorescent-quenching aptamers. Anal. Chem. 2023; 95(30):11399-409. DOI: 10.1021/acs.analchem.3c01577.
Chen W., Xu D., Liu L., Peng C., Zhu Y., Ma W. et al. Ultrasensitive detection of trace protein by Western blot based on POLY-quantum dot probes. Anal. Chem. 2009; 81(21):9194-8. DOI: 10.1021/ac901429a.
Hughes A.J., Herr A.E. Microfluidic Western blotting. Proc. Natl. Acad. Sci. U S A. 2012; 109(52):21450-5. DOI: 10.1073/pnas.1207754110.
Pan W., Chen W., Jiang X. Microfluidic Western blot. Anal. Chem. 2010; 82(10):3974-6. DOI: 10.1021/ac1000493.
Snowden B.W., Halliburton I.W. Identification of cross-reacting glycoproteins of four herpesviruses by Western blotting. J. Gen. Virol. 1985; 66 (Pt 9):2039-44. DOI: 10.1099/0022-1317-66-9-2039.
Begum H., Murugesan P., Tangutur A.D. Western blotting: a powerful staple in scientific and biomedical research. Biotechniques. 2022; 73(1):58-69. DOI: 10.2144/btn-2022-0003.
Horinouchi T., Terada K., Higashi T., Miwa S. Using phos-tag in Western blotting analysis to evaluate protein phosphorylation. Methods Mol. Biol. 2016; 1397:267-77. DOI: 10.1007/978-1-4939-3353-2_18.
Rigby L., Muscat A., Ashley D., Algar E. Methods for the analysis of histone H3 and H4 acetylation in blood. Epigenetics. 2012; 7(8):875-82. DOI: 10.4161/epi.2098.
Weekes J., Morrison K., Mullen A., Wait R., Barton P., Dunn M.J. Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics. 2003; 3(2):208-16. DOI: 10.1002/pmic.200390029.
Peng Z., Li X.J., Pang C., Zhang J.T., Zhu Q., Sun J.Q. et al. Hydrogen inhalation therapy regulates lactic acid metabolism following subarachnoid hemorrhage through the HIF-1α pathway. Biochem. Biophys. Res. Commun. 2023 Jun 30; 663:192-201. DOI: 10.1016/j.bbrc.2023.04.072.
Meftahi G.H., Bahari Z., Zarei Mahmoudabadi A., Iman M., Jangravi Z. Applications of Western blot technique: From bench to bedside. Biochem. Mol. Biol. Educ. 2021; 49(4):509-17. DOI: 10.1002/bmb.21516.
Owen C., Fader K.A., Hassanein M. Western blotting: evolution of an old analytical method to a new quantitative tool for biomarker measurements. Bioanalysis. 2024; 16(5):319-28. DOI: 10.4155/bio-2023-0212.
Garczyk S., von Stillfried S., Antonopoulos W., Hartmann A., Schrauder M.G., Fasching P.A. et al. AGR3 in breast cancer: prognostic impact and suitable serum-based biomarker for early cancer detection. PLoS One. 2015 Apr 15; 10(4):e0122106. DOI: 10.1371/journal.pone.0122106.
Guo Z., Li Y., Li W., Li H., Wu Z. Exosome-mediated lncRNA LINC01140 attenuates breast cancer progression by regulating the Wnt/β-Catenin Pathway. Crit. Rev. Eukaryot. Gene Expr. 2023; 33(7):31-42. DOI: 10.1615/CritRevEukaryotGeneExpr.2023048344.
Ali A., Kulik G. Signaling pathways that control apoptosis in prostate cancer. Cancers (Basel). 2021 Feb 24; 13(5):937. DOI: 10.3390/cancers13050937.
Tong G., Cheng B., Wu X., He L., Lv G., Wang S. circHIPK2 has a potentially important clinical significance in colorectal cancer progression via HSP90 ubiquitination by miR485-5p. Crit. Rev. Eukaryot. Gene Expr. 2022; 32(8):33-42. DOI: 10.1615/CritRevEukaryotGeneExpr.2022042925.
Kusuma V.I., I’tishom R., Qurnianingsih E., Rejeki P.S. A Systematical review of the effect of ketogenic diet on Bcl-2 (B-cell lymphoma-2) expression as an apoptosis marker in cancer treatment. Biomol. Health Sci. J. 2021; 4(2): 125–30. DOI: 10.20473/bhsj.v4i2.30173.
Jiménez C., Garrote-de-Barros A., López-Portugués C., Hernández-Sánchez M., Díez P. Characterization of human B cell hematological malignancies using protein-based approaches. Int. J. Mol. Sci. 2024; 25(9):4644. DOI: 10.3390/ijms25094644.
Monfaredan A., Rahim F., Tavoosidana G., Modarressi M.H., Hosseininasab A., Aghajani-Afrouzi A.A. et al. Imatinib loaded body fluids derived exosomes using microfluidics system: an in vitro analysis. Sys. Rev. Pharm. 2024; 15(3): 110-9. DOI: 10.31858/0975-8453.15.3.110-119.
Abdulkhaleq F., Larossi N., Ogbonda O., Abu Eid R., Ward F. CTLA-4 expression by human tumor cells and its impact on immunotherapeutic strategies: a systematic review. Immuno-Oncology Insights. 2024; 2(3):151-69. DOI: 10.18609/ioi.2021.024.
Sadeghzadeh J., Shahabi P., Farhoudi M., Ebrahimi-Kalan A., Mobed A., Shahpasand K. et al. Tau protein biosensors in the diagnosis of neurodegenerative diseases. Adv. Pharm. Bull. 2023; 13(3):502-11. DOI: 10.34172/apb.2023.061.
Estaun-Panzano J., Arotcarena M.L., Bezard E. Monitoring α-synuclein aggregation. Neurobiol. Dis. 2023; 176:105966. DOI: 10.1016/j.nbd.2022.105966.
Worrall D., Ayoubi R., Fotouhi M., Southern K., McPherson P.S., Laflamme C. et al. The identification of high-performing antibodies for TDP-43 for use in Western blot, immunoprecipitation and immunofluorescence. F1000Res. 2023 Jun 20; 12:277. DOI: 10.12688/f1000research.131852.2.
Gong P., Jia H.Y., Li R., Ma Z., Si M., Qian C. et al. Downregulation of Nogo-B ameliorates cerebral ischemia/reperfusion injury in mice through regulating microglia polarization via TLR4/NF-kappaB pathway. Neurochem. Int. 2023 Jul; 167:105553. DOI: 10.1016/j.neuint.2023.105553.
Maruyama R., Yokota T. Antisense oligonucleotide treatment in a humanized mouse model of Duchenne muscular dystrophy and highly sensitive detection of dystrophin using Western blotting. Methods Mol. Biol. 2021; 2224:203-14. DOI: 10.1007/978-1-0716-1008-4_15.
Sawai S., Mori M., Kuwabara S. Methodology for identification of new target molecules in neuroimmunological disorders. Clin. Exper. Neuroimmunol. 2021; 12(3): 202-7. DOI: 10.1111/cen3.12645.
Kumar N.N., Ahmad Dit. Al Hakim S., Grygiel-Górniak B. Antinuclear antibodies in non-rheumatic diseases. Arch. Immunol. Ther. Exp. (Warsz). 2025; 73(1): 10.2478/aite-2025-0004. DOI: 10.2478/aite-2025-0004.
Banerjee A., Goel A., Shah A., Srivastava S. Recent advances in proteomics and its implications in pituitary endocrine disorders. Biochim. Biophyos. Acta Proteins Proteom. 2021; 1869(11):140700. DOI: 10.1016/j.bbapap.2021.140700.
Kim K., Kwon J.S., Ahn C., Jeung E.B. Endocrine-Disrupting Chemicals and Their Adverse Effects on the Endoplasmic Reticulum. Int. J. Mol. Sci. 2022; 23(3):1581. Published 2022 Jan 29. DOI: 10.3390/ijms23031581.
Hassanabad A.F., Deniset J.F., Fedak P.W.M. Pericardial inflammatory mediators that can drive postoperative atrial fibrillation in cardiac surgery patients. Can. J. Cardiol. 2023; 39(8):1090-1102. DOI: 10.1016/j.cjca.2023.06.003.
Kawase Y., Shimizu K., Funamoto M., Sunagawa Y., Katanasaka Y., Miyazaki Y. et al. Young investigator award: compound A, a Ginger extract, significantly reduces pressure overload-induced systolic heart failure in mice. Eur. Cardiol. 2021; 16:e5. DOI: 10.15420/ecr.2021.16.PO1.
Dong Y., Zhou G., Cao W., Xu X., Zhang Y., Ji Z. et al. Global seroprevalence and sociodemographic characteristics of Borrelia burgdorferi sensu lato in human populations: a systematic review and meta-analysis. BMJ Glob. Health. 2022; 7(6):e007744. DOI: 10.1136/bmjgh-2021-007744.
Elschner M.C., Scholz H.C., Melzer F., Saqib M., Marten P., Rassbach A. et al. Use of a Western blot technique for the serodiagnosis of glanders. BMC Vet. Res. 2011 Jan 19; 7:4. DOI: 10.1186/1746-6148-7-4.
Bass J.J., Wilkinson D.J., Rankin D., Phillips B.E., Szewczyk N.J., Smith K. et al. An overview of technical considerations for Western blotting applications to physiological research. Scand. J. Med. Sci. Sports. 2017; 27(1):4-25. DOI: 10.1111/sms.12702.