Abstract
An ultra-rapid method for electrophoresing proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to nitrocellulose membranes, and immunoblotting is described here. Electrophoresis of the autoantigens La and Ro60, as well as molecular weight standards on a 4–20 % gradient gel, was performed in about 10 min using heated (70–75 °C) normal Laemmli running buffer. Electrophoretic transfer of these proteins was achieved in 7 min using a semidry transfer method. Finally, immunoblotting of La and Ro60 was carried out in 30 min. Thus, the entire process of electrophoresis, electrotransfer, and immunoblotting could be carried out in 1 h.
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References
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Srinivas PR (2012) Introduction to protein electrophoresis. Methods Mol Biol 869:23–28
Haeberle JR (1997) High-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis. Biotechniques 23:638–640
Kurien BT, Scofield RH (1997) Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 205:91–94
Peferoen M, Huybrechts R, De Loof A (1982) Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to NC. FEBS Lett 145:369–372
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Proc Natl Acad Sci U S A 76:4350–4354
Bolt MW, Mahoney PA (1997) High efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 247:185–192
MacPhee DJ (2010) Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods 61:171–177, Review
Biorad transblot turbo transfer system. http://www.bio-rad.com/en-us/product/semi-dry-rapid-blotting-systems/trans-blot-turbo-transfer-system
Kurien BT, Scofield RH (2000) Ultrarapid electrophoretic transfer of high and low molecular weight proteins using heat. Methods Mol Biol 536:181–190
Kurien BT, Scofield RH (2002) Heat-mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes. J Immunol Methods 266:127–133
Huang SC, Pan Z, Kurien BT, James JA, Harley JB, Scofield RH (1995) Immunization with vesicular stomatitis virus nucleocapsid protein induces autoantibodies to the 60 kD Ro ribonucleoprotein particle. J Investig Med 43:151–158
Kurien BT, Asfa S, Li C, Dorri Y, Jonsson R, Scofield RH (2005) Induction of oral tolerance in experimental Sjogren’s syndrome autoimmunity. Scand J Immunol 61:418–425
Tröster H, Bartsch H, Klein R, Metzger TE, Pollak G, Semsei I, Schwemmle M, Pruijn GJ, van Venrooij WJ, Bachmann M (1995) Activation of a murine autoreactive B cell by immunization with human recombinant autoantigen La/SS-B: characterization of the autoepitope. J Autoimmun 8:825–842
DenHollander N, Befus D (1989) Loss of antigens from immunoblotting membranes. J Immunol Methods 122:129–135
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Kurien, B.T., Danda, D., Bachmann, M.P., Scofield, R.H. (2015). SDS-PAGE to Immunoblot in One Hour. In: Kurien, B., Scofield, R. (eds) Western Blotting. Methods in Molecular Biology, vol 1312. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2694-7_45
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DOI: https://doi.org/10.1007/978-1-4939-2694-7_45
Publisher Name: Humana Press, New York, NY
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