Non-radioactive labelling and immunoprecipitation analysis of leukocyte surface proteins using different methods of protein biotinylation.
The biotinylation of surface proteins and the detection of immunoprecipitated protein(s) after transfer to nitrocellulose using chemiluminescence methods is a highly sensitive alternative to hazardous radioactive labelling procedures. Ligation of proteins using the common biotin-NHS-ester (N-hydroxysuccinimido-biotin) is often associated with a decrease in immunoreactivity. Here a new non-radioactive method of leukocyte surface glycoprotein labelling using biotin-LC-hydrazide is described. This technique is based on the labelling of glycoproteins after mild oxidation of carbohydrate hydroxyl groups to reactive aldehydes. Flow cytometric and immunoprecipitation analyses of selected leukocyte markers such as CD3, CD26 and CD65 indicated that the alteration in immunoreactivity achieved by NHS-mediated biotin ligation was different from that obtained with hydrazide-mediated biotin ligation. CD3 and CD26 immunoreactivity was diminished using NHS biotinylation but preserved by biotin-LC-hydrazide, whereas CD65 binding to monoclonal antibodies was completely abolished after treatment with biotin-hydrazide. However, the immunoreactivity of CD13 was found to be totally unaffected by both NHS and hydrazide biotinylation. The combination of different biotinylation methods for surface protein labelling offers a viable alternative to radioiodination in the biochemical analysis of membrane proteins.
Kähne
T
T
Medical Academy Magdeburg, Department of Internal Medicine, Germany.
Ansorge
S
S
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology; D001710:Biotin / Q000031:analogs & derivatives; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008565:Membrane Proteins / Q000032:analysis*; D011233:Precipitin Tests / Q000379:methods*; D013388:Succinimides; D013601:T-Lymphocytes / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D008565:Membrane Proteins; D013388:Succinimides; C033788:biotinyl N-hydroxysuccinimide ester; C053311:biotin hydrazide; D001710:Biotin