Dear experts, I am trying to couple a derivative of BSA to antibodies. The BSA has blocked amino groups. I activate the carboxyls for 15min at RT with EDC (1mg EDC/1mg BSA) and NHS in 20mM MES pH 5, then remove excess carbodiimide with 100 µl of an ion exchanger (carboxylic groups, amberlyst CNP105/H+ form, previously equilibrated in MES), then add the BSA in 5 or 10x molar excess to the antibody which is 1mg at 1mg/ml in 200mM Phosphate, pH 8. After 2 hrs I quench with 100µl 1M Tris pH 7.5.
I test the product with an ELISA (coated antigen, detection with antibody- HRP-conjugate against something that has been coupled to the lysines of the BSA before). I get almost no signal. The antibody is functional after the reaction and exactly binding like the untreated control (detected by anti-species-HRP). I suppose that there is (almost) no coupling at all, as then, in an analytical gel filtration (Sephacryl S 300, 60cm column), the antibody is the largest molecule that I can see. Can you perhaps give a comment on the EDC chemistry? Basically, I am using a protocol given by Pierce for their crosslinking kits. Any idea how to test if the EDC has gone bad? (no access to IR and NMR, however) Is activation/coupling time too short? Do I maybe need a spacer? Any other possibility of removing excess EDC (am already thinking of gel filtration with a PD10 column)? Mercaptoethanol (as suggested in the original protocol) is probably not an option, as leftovers of BME are likely to kill the antibody. Any ideas/suggestions/comments? Thanks for your help! Wo _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
