I have a question for those doing immunos by hand. I typically dilute my primary, secondary and tertiary antibodies with manufacturer supplied diluent. Recently, I was running a test to determine the source of a mysterious precipitate that had begun to appear on my slides (It has since then disappeared just as mysteriously). In one of the conditions, I substituted the diluent with TBST for all dilutions. I found that this particular slide had noticeably less background. When inquiring as to what I should be diluting these reagents with, I received conflicting answers from tech support. I heard that the secondary and tertiary antibodies should be diluted with what they are actually in, i.e. .05% TBS or 1M PBS. I also was told that I could dilute everything with diluent.
Can anyone tell me which is correct? Or why using the buffer yielded less background than the diluent? Aside from sodium azide and BSA I don't know what's in the proprietary reagent. This communication is intended solely for the use of the addressee and may contain information that is legally privileged, confidential or exempt from disclosure. If you are not the intended recipient, please note that any dissemination, distribution, or copying of this communication is strictly prohibited. Anyone who receives this message in error should notify the sender immediately and delete it from his or her computer _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet