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:: Volume 10, Issue 2 (Spring 2022) ::
Shefaye Khatam 2022, 10(2): 133-148 Back to browse issues page
Western Blotting Protocol and its Troubleshooting
Javad Momeni , Sajad Sahab Negah *
a. Neuroscience Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. b. Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran , sahabnegahs@mums.ac.ir
Abstract:   (2340 Views)
Introduction: The detection of protein expression in tissues and nerve cells is necessary for the modern neuroscience research laboratory and laboratories making diagnostic approaches. To this point, we collected a comprehensive review article on steps and troubleshooting of Western blotting (WB). WB provides good qualitative results for determining a sample containing a target protein and is particularly useful for analyzing insoluble proteins. This process basically can be divided into five steps: (I) the protein extraction, (II) electrophoresis, (III) membrane transfer, (IV) immunodetection, and (V) visualization and analysis. These processes involve the transfer of protein patterns from gel to microporous membrane. Protein transfer with subsequent immunodetection is a great tool to identify a multitude of proteins, especially those proteins that are of low abundance. The efficient transfer of proteins from a gel to a solid membrane and specific immunodetection depend greatly on different parameters. Conclusion: For reducing the troubleshooting and enhancing awareness of the new developments in WB, this review summaries a representative of methods, reagents, and devices in the WB procedure.
Keywords: Neurosciences, Laboratories, Immunoblotting
Full-Text [PDF 2990 kb]   (3260 Downloads)    
Type of Study: Review --- Open Access, CC-BY-NC | Subject: Basic research in Neuroscience
attachement [PDF 1149 KB]  (89 Download)
1. Southern EM. 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]
2. Alwine JC, Kemp DJ, Stark GR. 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(12): 5350-4. [DOI:10.1073/pnas.74.12.5350]
3. 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. 1981; 112(2): 195-203. [DOI:10.1016/0003-2697(81)90281-5]
4. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America. 1979; 76(9): 4350-4. [DOI:10.1073/pnas.76.9.4350]
5. Bass JJ, Wilkinson DJ, Rankin D, Phillips BE, Szewczyk NJ, Smith K, et al. An overview of technical considerations for Western blotting applications to physiological research. Scandinavian Journal of Medicine & Science in Sports. 2017; 27(1): 4-25. [DOI:10.1111/sms.12702]
6. Jalali Kondori B, Asadi MH, Azemati F. Subcloning of NGF Gene into pSecTag2/Hygro Secretory Vector and Expression in PC12 Cell Line. The Neuroscience Journal of Shefaye Khatam. 2015; 3(1): 21-8. [DOI:10.18869/acadpub.shefa.3.1.21]
7. Mehrabi A, Gaeini A, Nouri R, Daryanoosh F. The Effect of Six-Week HIIT Swimming Exercise and Resveratrol Supplementation on the Level of SIRT3 in Frontal Lobe of Aged Rats. The Neuroscience Journal of Shefaye Khatam. 2021; 9(2): 48-59. [DOI:10.52547/shefa.9.2.48]
8. Najmi Z, Ghasemi S, Ghalandari R, Sotoudehnejad Nematalahi F. Production and Evaluation of Anti-Mouse Polyclonal Antibody Against Enterotoxin B of Staphylococcus Aurous. The Neuroscience Journal of Shefaye Khatam. 2020; 8(2): 82-92. [DOI:10.29252/shefa.8.2.82]
9. Li R, Shen Y. An old method facing a new challenge: re-visiting housekeeping proteins as internal reference control for neuroscience research. Life Sci. 2013; 92(13): 747-51. [DOI:10.1016/j.lfs.2013.02.014]
10. Carter M, Shieh JC. Chapter 14 - Biochemical Assays and Intracellular Signaling. In: Carter M, Shieh JC, editors. Guide to Research Techniques in Neuroscience. New York: Academic Press; 2010. p. 297-329. [DOI:10.1016/B978-0-12-374849-2.00014-8]
11. Doumas BT, Bayse DD, Carter RJ, Peters T, Jr., Schaffer R. A candidate reference method for determination of total protein in serum. I. Development and validation. Clin Chem. 1981; 27(10): 1642-50. [DOI:10.1093/clinchem/27.10.1642]
12. Mahmood T, Yang P-C. Western blot: technique, theory, and trouble shooting. N Am J Med Sci. 2012; 4(9): 429-34. [DOI:10.4103/1947-2714.100998]
13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54. [DOI:10.1016/0003-2697(76)90527-3]
14. Desjardins P, Hansen JB, Allen M. Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer. JoVE (Journal of Visualized Experiments). 2009; (33): e1610. [DOI:10.3791/1610]
15. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259): 680-5. [DOI:10.1038/227680a0]
16. Anfinsen CB. Principles that govern the folding of protein chains. Science. 1973; 181(4096): 223-30. [DOI:10.1126/science.181.4096.223]
17. Krieg RC, Dong Y, Schwamborn K, Knuechel R. Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE. J Biochem Biophys Methods. 2005; 65(1): 13-9. [DOI:10.1016/j.jbbm.2005.08.005]
18. Smith BJ. SDS Polyacrylamide Gel Electrophoresis of Proteins. Methods Mol Biol. 1984; 1: 41-55. [DOI:10.1385/0-89603-062-8:41]
19. Taylor SC, Posch A. The design of a quantitative western blot experiment. Biomed Res Int. 2014; 2014: 361590. [DOI:10.1155/2014/361590]
20. Weber K, Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969; 244(16): 4406-12. [DOI:10.1016/S0021-9258(18)94333-4]
21. Ornstein L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964; 121: 321-49. [DOI:10.1111/j.1749-6632.1964.tb14207.x]
22. Chrambach A, Rodbard D. Polyacrylamide gel electrophoresis. Science. 1971; 172(3982): 440-51. [DOI:10.1126/science.172.3982.440]
23. Rath A, Cunningham F, Deber CM. Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts. Proc Natl Acad Sci U S A. 2013; 110(39): 15668-73. [DOI:10.1073/pnas.1311305110]
24. Warren CM, Krzesinski PR, Greaser ML. Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins. Electrophoresis. 2003; 24(11): 1695-702. [DOI:10.1002/elps.200305392]
25. Walker JM. Gradient SDS polyacrylamide gel electrophoresis of proteins. Methods Mol Biol. 1994; 32: 35-8. [DOI:10.1385/0-89603-268-X:35]
26. Kurien BT, Scofield RH. Western blotting. Methods. 2006; 38(4): 283-93. [DOI:10.1016/j.ymeth.2005.11.007]
27. MacPhee DJ. Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods. 2010; 61(2): 171-7. [DOI:10.1016/j.vascn.2009.12.001]
28. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987; 262(21): 10035-8. [DOI:10.1016/S0021-9258(18)61070-1]
29. Mansfield MA. Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking. Anal Biochem. 1995; 229(1): 140-3. [DOI:10.1006/abio.1995.1391]
30. Mozdzanowski J, Hembach P, Speicher DW. High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels. Electrophoresis. 1992; 13(1-2): 59-64. [DOI:10.1002/elps.1150130112]
31. 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]
32. Colella AD, Chegenii N, Tea MN, Gibbins IL, Williams KA, Chataway TK. Comparison of Stain-Free gels with traditional immunoblot loading control methodology. Anal Biochem. 2012; 430(2): 108-10. [DOI:10.1016/j.ab.2012.08.015]
33. Kurien BT, Danda D, Bachmann MP, Scofield RH. SDS-PAGE to Immunoblot in One Hour. Methods Mol Biol. 2015; 1312: 449-54. [DOI:10.1007/978-1-4939-2694-7_45]
34. Jensen EC. The basics of western blotting. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology. 2012; 295(3): 369-71. [DOI:10.1002/ar.22424]
35. Spinola SM, Cannon JG. Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes. Journal of immunological methods. 1985; 81(1): 161-5. [DOI:10.1016/0022-1759(85)90132-2]
36. Gershoni JM, Palade GE. Protein blotting: principles and applications. Analytical biochemistry. 1983; 131(1): 1-15. [DOI:10.1016/0003-2697(83)90128-8]
37. Kurien BT, Dorri Y, Dillon S, Dsouza A, Scofield RH. An overview of Western blotting for determining antibody specificities for immunohistochemistry. Signal Transduction Immunohistochemistry. 2011: 55-67. [DOI:10.1007/978-1-61779-024-9_3]
38. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F. Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR journal. 2005; 46(3): 258-68. [DOI:10.1093/ilar.46.3.258]
39. Voller A, Bartlett A, Bidwell D. Enzyme immunoassays with special reference to ELISA techniques. Journal of clinical pathology. 1978; 31(6): 507-20. [DOI:10.1136/jcp.31.6.507]
40. Miura T, Suzuki W, Ishihara E, Arai I, Ishida H, Seino Y, et al. Impairment of insulin-stimulated GLUT4 translocation in skeletal muscle and adipose tissue in the Tsumura Suzuki obese diabetic mouse: a new genetic animal model of type 2 diabetes. European journal of endocrinology. 2001; 145(6): 785-90. [DOI:10.1530/eje.0.1450785]
41. Crossland H, Kazi AA, Lang CH, Timmons JA, Pierre P, Wilkinson DJ, et al. Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway. American Journal of Physiology-Endocrinology and Metabolism. 2013. [DOI:10.1152/ajpendo.00541.2012]
42. Bronstein I, Voyta J, Thorpe G, Kricka L, Armstrong G. Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin. Clinical chemistry. 1989; 35(7): 1441-6. [DOI:10.1093/clinchem/35.7.1441]
43. Kricka LJ. Chemiluminescent and bioluminescent techniques. Clinical chemistry. 1991; 37(9): 1472-81. [DOI:10.1093/clinchem/37.9.1472]
44. Babson AL, Greeley SJ, Coleman CM, Phillips GE. Phenolphthalein monophosphate as a substrate for serum alkaline phosphatase. Clinical chemistry. 1966; 12(8): 482-90. [DOI:10.1093/clinchem/12.8.482]
45. Alegria-Schaffer A, Lodge A, Vattem K. Performing and optimizing Western blots with an emphasis on chemiluminescent detection. Methods in enzymology. 2009; 463: 573-99. [DOI:10.1016/S0076-6879(09)63033-0]
46. Argentieri MC, Pilla D, Vanzati A, Lonardi S, Facchetti F, Doglioni C, et al. Antibodies are forever: a study using 12-26‐year‐old expired antibodies. Histopathology. 2013; 63(6): 869-76. [DOI:10.1111/his.12225]

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Momeni J, Sahab Negah S. Western Blotting Protocol and its Troubleshooting. Shefaye Khatam 2022; 10 (2) :133-148
URL: http://shefayekhatam.ir/article-1-2241-en.html

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Volume 10, Issue 2 (Spring 2022) Back to browse issues page
مجله علوم اعصاب شفای خاتم The Neuroscience Journal of Shefaye Khatam
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