Efficient radioiodination and immunopurification of surface proteins from small numbers of cells.
This report describes a method for the cell surface radioiodination and immunoprecipitation of the T cell receptor from as few as 7.5 x 10(4) lymphoid cells. The membranes of cells were labelled using the lactoperoxidase/H2O2 method. In order to minimize the loss of labelled cells after iodination, T cell receptor-negative 'filler' cells were added to the washes and the washing volume was kept small. Before immunoprecipitation, the solid-phase Staphylococcus aureus was precoated with the specific monoclonal antibody to ensure optimal recovery of antibody-antigen complexes. These simple modifications greatly increased the efficiency of the iodination and immunoprecipitation procedures, thus enabling direct analysis of membrane proteins from small numbers of cells.
Pearse
M J
MJ
Walter & Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.
Gallagher
P F
PF
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis; D002460:Cell Line; D002469:Cell Separation; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D007457:Iodine Radioisotopes; D007784:Lactoperoxidase / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis*; D011233:Precipitin Tests; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013205:Staphylococcal Protein A / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D013205:Staphylococcal Protein A; D007784:Lactoperoxidase