SpringerProtocols: Abstract: Nonelectrophoretic Bidirectional Transfer of a Single SDS-PAGE Gel with Multiple Antigens to Obtain 12 Immunoblots

8. Nonelectrophoretic Bidirectional Transfer of a Single SDS-PAGE Gel with Multiple Antigens to Obtain 12 Immunoblots

By: Biji T. Kurien²

, R. Hal Scofield²

Abstract

Full Text

Download PDF (265K)

Protein blotting is an invaluable technique in immunology to detect and characterize proteins of low abundance. Proteins resolved on sodium dodecyl sulfate (SDS) polyacrylamide gels are normally transferred electrophoretically to adsorbent membranes such as nitrocellulose or polyvinylidene diflouride membranes. Here, we describe the nonelectrophroretic transfer of the Ro 60 (or SSA) autoantigen, 220- and 240-kD spectrin antigens, and prestained molecular weight standards from SDS polyacrylamide gels to obtain up to 12 immunoblots from a single gel and multiple sera.

Affiliation(s): (2) Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104, USA

Book Title: Protein Blotting and Detection: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 536 | Pub. Date: Feb-06-2009 | Page Range: 55-65 | DOI: 10.1007/978-1-59745-542-8_8

Subject: Protein Science

Key Words: Nonelectrophoretic transfer - Immunoblots - Bidirectional transfer - Nitrocellulose membrane - Autoantibodies - Autoantigens

References

Download References

1.	Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Proc Natl Acad Sci USA 76, 4350–4354.

2.	Kurien, B.T. and Scofield, R.H. (2006) Western blotting. Methods 38, 283–293.

3.	Renart, J., Reiser, J., and Stark, G.R. (1979) Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with anti-sera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci USA 76, 3116–3120.

4.	Gershoni, J.M. and Palade, G.E. (1983) Protein blotting: principles and applications. Anal Biochem 131, 1–15.

5.	Peferoen, M., Huybrechts, R., and 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.

6.	Kurien, B.T. and Scofield, R.H. (1997) Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 205, 91–94.

7.	Smith, G.E. and Summers, M.D. (1980) The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem 109, 123–129.

8.	Reinhart, M.P. and Malamud, D. (1982) Protein transfer from isoelectric focusing gels: the native blot. Anal Biochem 123, 229–235.

9.	Knisley, K.A. and Rodkey, L.S. (1986) Affinity immunoblotting. High resolution isoelectric focusing analysis of antibody clonotype distribution. J Immunol Methods 95, 79–87.

10.	Scofield, R.H., Zhang, F., Kurien, B.T., Anderson, C.J., Reichlin, M., Harley, J.B.,(1996) Development of the anti-Ro autoantibody response in a patient with systemic lupus erythematosus. Arthritis Rheum 39, 1664–1668.

11.	Deutscher, S.L., Harley, J.B., and Keene, J.D. (1988) Molecular analysis of the 60 kD human Ro ribonucleoprotein. Proc Natl Acad Sci USA 85, 9479–9483.

12.	Ben-Chetrit, E., Gandy, B.J., Tan, E.M., and Sullivan, K.F. (1989) Isolation and characterization of a cDNA clone encoding the 60 kDa component of the human SS-A/Ro ribonucleoprotein autoantigen. J Clin Invest 83, 1284–1292.

13.	Dodge, J.T., Mitchell, C., and Hanahan, D.J. (1963) The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys 100, 119–130.

14.	Harlow, E., and Lane, D. (1988) Electrophoresis. Appendix I. In: Harlow, E. and Lane, D. Ed. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York, p. 638.

15.	Harlow, E., and Lane, D. (1988) Electrophoresis. Appendix I. In: Harlow, E. and Lane, D. Ed. Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, New York, p. 636.

16.	Wu, M., Stockley, P.G., and Martin, W.J. II (2002) An improved Western blotting technique effectively reduces background. Electrophoresis 23, 2373–2376.

Comments (Loading...)

Post comment/View all comments