WB trouble - no signal of low kd protein - (Oct/17/2007 )

hello


my wb is cutted in different parts for different antibodies.

i see phospho antibodies at around 60 kd, but suddendly all my lower protein at 36 kd show no signal anymore.

my conditions are the same, and the total of this protein is not showing up either.

bsa 5% for blocking at rt for 2hours and the primary antibody 1:1000 in 1% bsa overnight
then 4x 5 minutes washes, secondary is anti rabbit 1:5000 for 45 minutes
4x 5 min washes and my signal of all the other proteins from the same membrane are fine,,,,,

i need help!!!!

thank you franzi

against what is your Ab directed? Anti-Phospho-Y or -S-/-T? or against a distinct phospho-polypeptide? did you check abundance of polypeptides <36 kDa, f.i. by Ponceau S stain?



my antibody is a phospho- ser antibody.
i stained my membrane with coomassie and i could see bands.

Even if you see bands, it might happen that most of your low MW proteins are passing through the membrane... Try placing two membranes together, and stain them with Ponceau after the transfer is done. If your setup is OK you shouldn't see any bands in the second membrane...

CBB is an irreversible stain and should not proceed immunostain on the same blot
[/quote]


i know that cbb is an irreversible stain...but i have tried this blot for several times now,
always same conditions

one of them i stained with cbb



the primary antibody is ok, because i ordered new one
the secondary is ok, because i get a signal with another protein....

i am devasted

thx