Purification of the T cell antigen receptor and analysis by blue-native PAGE.
The T cell antigen receptor (TCR) is a multi-protein complex composed of six different transmembrane subunits, which form complexes of various sizes on the surface of resting T cells. The stoichiometry of the smallest form was recently determined to be alphabetagammaepsilondeltaepsilonzetazeta, whereas that of the larger forms is unknown. The roles of the different forms and their ratios are poorly defined. Biochemical analyses to address these questions must focus on the detergent and the best native conditions to maintain the integrity of the complexes. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a high-resolution native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation. Using this powerful approach, the size, subunit composition and the relative abundance of the different TCR forms can be studied. We present here four methods to isolate the TCR in a native form and details to analyse it by BN-PAGE.
Swamy
Mahima
M
Department of Molecular Immunology, Max Planck Institute for Immunobiology, University of Freiburg, Freiburg, Germany.
Schamel
Wolfgang W A
WW
D000818:Animals; D045163:Antibodies, Phospho-Specific / Q000276:immunology; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012394:Rosaniline Dyes
D045163:Antibodies, Phospho-Specific; D021122:Protein Subunits; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D012394:Rosaniline Dyes; C048139:Coomassie blue