cathepsin C detection in Western blot - (Aug/03/2007 )
a colleague of mine is trying to detect Cathepsin C in blood leukocytes using primary polyclonal antibodies (anti- cathepsin C H-144: sc-13986 from Santa Cruz Biotechnology Inc.). The samples were prepared under reducing and non-reducing conditions. Oddly enough she is getting distinct bands between 50 and 60 kDa in lanes where only sample buffer containing DTT or B-ME but no Cathepsin C was loaded (negative control). Another odd thing was that also the MW Marker Prestained Precision Plus from BioRad showed several bands across the entire lane (it also contains 10 mM DTT). We are aware that polyclonal primary antibodies can bind to a number of different things but binding to DTT seems a bit strange. Secondary antibodies showed no signal with the same samples. Has anyone else experienced a similar thing and what could be the reason for it?
Thanks for your help,
these bands are caused by reducing agent and dust. they have been potentially identified as keratins. they seem to exhibit non-specific binding in western blots (i'm not sure if it is with the primary or secondary antibody or both). there have been attempts to reduce the artifacts by using fresher reducing agent, addition of sodium metabisulfite, etc. we have found that the best way to avoid the bands is to eliminate the reducing agent from the gel but this can not be done for all proteins.