multiple bands from monoclonal antibody - (Sep/27/2004 )

Hi!

I'm raising monoclonal antibody against HN and F protein from Newcastle Disease Virus (NDV). I did cloning three times (single cell per well). When I did my western blot against my antigen (whole virus), I managed to get multiple bands (maybe unspecific bands?). Should I get single band only? Thanks.

Andrew.

don't worry that's perfectly normal!

as you mentioned yourself, the bands you see are most likely unspecific crossreactivity of your ab. for western blotting you just have to find the right concentration for you ab to show only the band your interested in. so you should use less ab and see what's happening.

the other possibility is, that there are the same or very similiar epitopes on other proteins than the one you're interested in. since abs recognise streches of 9-14 aminoacids, it can happen that the same or similar streches are found in other proteins, too, that will be consequently recognised by your ab.

BTW, have you thought of different isoforms of you protein? spliceforms or heavily modificated prodeins do run at different sizes.

to check the monoclonality of your ab you should do IgG isoform determination (i.e. is it IgG1, IgG2, IgG3 etc.). that should be stable over time. the only way to really be sure if it's a mAb, you have to do some epitopemapping, and if it's truly a mAb, it should bind to only one, determined epitope.

mike