Crossreactivity or protein complex under denaturing conditions? - (Mar/08/2008 )
Hello everyone,
I would appreciate some brainstorming with this awkward thing I came across :
I am studying protein A (69kDa) and protein B (10.5kDa) in two different projects by doing Western blots. Quite coincidentaly I discovered that when I incubate two membranes (same samples in both) with antibodies against those 2 proteins (one on each membrane of course!) I get the same band at about 80kDa according to my protein standard. The antibodies are (should be) monoclonal from Sigma. So my question is :
a. Is it just crossreactivity between the antibodies, but if it is so, why do the bands appear in a somewhat higher molecular weight? A colleague of mine said that what I see as closer to 80kDa is probably about 70kDa in compliance with one of the two proteins.
b. The fact that I see the band closer to the 80kDa (69+10.5=79.5) makes it very convenient for me to believe that my proteins are somehow bound to each other. But can that happen when I run Western under denaturing conditions?? SDS and β-mercaptoethanol are included in my sample buffer!!
Thanks for the comments!
Ithace (....it's the journey that counts...not the destination!)
hi,
a quick question...have you probed the membranes w/ only your secondary antibody? it's a trivial explanation, but every now and then it's right, and therefore warrants testing.
also, do you know if these two proteins form a covalent linkage between each other (non-disulfide) (think Ubiqutin, SUMO, etc)?
how long are you boiling your extracts...sometimes proteins and peptides are just annoyingly sticky and require harsh measures to be separated. i don't recommend thinking of people in the same way.
lastly, do these proteins contain common motifs that would (in a very remote chance) also correspond to the epitopes against which the antibodies were raised?
I would appreciate some brainstorming with this awkward thing I came across :
I am studying protein A (69kDa) and protein B (10.5kDa) in two different projects by doing Western blots. Quite coincidentaly I discovered that when I incubate two membranes (same samples in both) with antibodies against those 2 proteins (one on each membrane of course!) I get the same band at about 80kDa according to my protein standard. The antibodies are (should be) monoclonal from Sigma. So my question is :
a. Is it just crossreactivity between the antibodies, but if it is so, why do the bands appear in a somewhat higher molecular weight? A colleague of mine said that what I see as closer to 80kDa is probably about 70kDa in compliance with one of the two proteins.
b. The fact that I see the band closer to the 80kDa (69+10.5=79.5) makes it very convenient for me to believe that my proteins are somehow bound to each other. But can that happen when I run Western under denaturing conditions?? SDS and β-mercaptoethanol are included in my sample buffer!!
Thanks for the comments!
Ithace (....it's the journey that counts...not the destination!)
Well....no, I haven't probed my membranes with secondary only. I have to admit I do not really get the idea here. Can it be so that my secondary antibody exhibits such a selectivity for whatever is there on that MW regardless of whether this "whatever" has a primary antibody on it or not?? I can check it out anyway, so thanks for the tip. As far as I know these two proteins have no association to each other whatsoever (although they both sit closely on 1q chromosome and that's the closest relation I can think of). About boiling my samples, I do it for 5 minutes. And as far as common motifs are concerned, I checked that already and I could not find any!!! Tricky, right?? At least to my eyes.....oh,well.....I am just an MD striving in the lab!!! Not really my field and not my cup of tea, if I may say so! Thanks for the help once again!
Ithace