Diagonal Gel Electrophoresis

L.E. Samelson1

1 National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.6
DOI:  10.1002/0471142735.im0806s02
Online Posting Date:  May, 2001
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Abstract

Diagonal gel electrophoresis is a form of two‐dimensional analysis useful for investigating the subunit composition of multisubunit proteins containing interchain disulfide bonds. Proteins are electrophoresed in the first dimension in a slab or tube gel under nonreducing conditions. The proteins are then reduced in the gel and this piece of gel is layered onto a second gel and electrophoresed. In the second gel, the proteins migrate at right angles to their original, first‐dimension migration. The majority of cellular proteins are not disulfide‐linked and will fall on the “diagonal” in this system; that is, they migrate approximately equal distances in both directions during electrophoresis and lie approximately on the diagonal line connecting opposite corners of the gel. Upon reduction, component subunits of proteins connected by interchain disulfide bonds will resolve below the diagonal because the individual subunits migrate faster than the disulfide‐linked complex during the second electrophoresis.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Separating and stacking gel solutions (unit 8.4)
  • 1× SDS/sample buffer without reducing agents (unit 8.4)
  • recipeReducing buffer
  • 1.5% agarose in recipereducing buffer
  • Syringe with a fine spinal needle (21‐ to 23‐G, 3‐in. long) 65°C water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4), two‐dimensional electrophoresis (unit 8.5), immunoprecipitation (unit 8.3), protein staining (unit 8.9), and autoradiography ( appendix 3A)
CAUTION: Acrylamide monomer is neurotoxic. Gloves should be worn when handling solutions, and the solution should not be pipetted by mouth.
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Figures

Videos

Literature Cited

Literature Cited
   Allison, J.A., McIntyre, B.W., and Bloch, D. 1982. Tumor‐specific antigen of murine T‐lymphoma defined with monoclonal antibody. J. Immunol. 129: 2293‐2300.
   Dulis, B.H., Kloppel, T.M., Grey, H.M., and Kubo, R.T. 1982. Regulation of catabolism of IgM heavy chains in a B lymphoma cell line. J. Biol. Chem. 257: 4369‐4374.
   Goding, J.W. and Harris, A.W. 1981. Subunit structure of cell surface proteins: Disulfide bonding in antigen receptors, Ly‐2/3 antigens, and transferrin receptors of murine T and B lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 76: 4530‐4534.
   Hannum, C.H., Kappler, J.W., Trowbridge, I.S., Marrack, P., and Freed, J.H. 1984. Immunoglobulin‐like nature of the alpha chain of a human T‐cell antigen/ MHC receptor. Nature (Lond.) 312: 65‐67.
   Sommer, A. and Traut, R.R. 1974. Diagonal polyacrylamide dodecyl sulfate gel electrophoresis for the identification of ribosomal proteins crosslinked with methyl‐4‐mercaptobutyrimidate. Proc. Natl. Acad. Sci. U.S.A. 71: 3946‐3950.
   Wang, K. and Richards, F.M. 1974. An approach to nearest neighbor analysis of membrane proteins. J. Biol. Chem. 249: 8005‐8018.
   Weissman, A.M., Samelson, L.E., and Klausner, R.D. 1986. A new subunit of the human T cell antigen receptor complex. Nature (Lond.) 312: 480‐482.
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