Problem: mouse antibodies with mouse tissues - (Mar/23/2012 )
Hi, i need help with this big problem.
As i refer in title i'm having problems in my western assays. i'm trying to detect my protein in mouse muscle extracts with a monoclonal mouse antibody (the only that exist against my glycosidic residues).
If I perform an assay with primary antibody and without primary, detect the same bands, so the secondary antibody is detecting nonspecific proteins. These bands appear at 100-150 KDa, i don't think that are mouse Immunoglobulins because the sample is boiled previously (but i don't know if possible).
I've tried to change percentage of tween-20 in wash buffer, different dilutions of primary and secondary antibody. I don't know that more i can change
Could someone help me?
check the data sheet that comes with the antibody (if it is commercial). see if it reacts with the mouse protein. if so then check to see if it's suitable for western blots (it may be against native protein only)
thanks for your reply
i've checked the data sheet and primary antibody has "cross-reactivity" with mouse protein (i don't know if this is that you refer). Also is recommeded for western blot.
I've just performed other assay in which i've changed the secondary antibody (from other commercial) and it seems that now i don't detect so much unspecific band. is it possible that?
absolutely. some antibodies are less specific than others.
you may be able to reduce background even more by preabsorbing the antibodies (both primary and secondary) with normal serum from the species in which the secondary is made. just add 2-5% in the antibody solutions.
ok! i'll try it for next westerns
thks so much for help me with this problem