Antibody not binding to low MW targets - (Mar/27/2015 )
I am try to blot for gluthathionylated proteins using Anti-PSSG Ab against total cell proteins. Ideally it should bind against all the proteins which are gluathionylated. Literature shows that proteins ranging from 200 to 20 Kda get glutathionylated.
But for me I am only getting few of bands of high MW proteins. Below that the bands just end abruptly without even the faintest signal. Only one time I got around 20 bands at different MWs. But after that I am not being able to get the same result. For some reason, the smaller and medium proteins are not shping glutathionylation at all. Only two bands at high MW.
Proteins are transferred properly as shown by Ponceau. Is it possible that smaller proteins are getting transferrd to the other side of the membrance?
Anybody can help?
can you post a picture of the blot, ponceau stained blot, stained gel after transfer, and, if possible, a parallel gel that hasn't been transferred?
if it was on the other side of the membrane you would still see it.
did the ponceau stained blot match the ponceau stained blot that worked as predicted?
Extremely sorry for the delay.
Protein were loaded at 20 ug and 40 ug respectively. Ponceu is not available at this moment. As you can see, the blot stops abruptly. Sorry, I don't have good quality image ATM.
it doesn't look like it stopped abruptly, more likely just didn't migrate very far.
what are your page conditions?
10% Gel with 4% stack, No DTT, 100 Volts Constant, Tris Glycine running buffer (with SDS). Ponceu showed that proteins resolved well but bands are restricted to that region.