Hey all,
I am new to WB and I am trying to detect the acetylated form of EP300/CBP but I get extremely faint bands and also almost a smear of unspecific bands.
I have tried leaving the Ab overnight and later on for only ~2h to minimise unspecif binding but nothing seems to improve my band of interest and the unspecific binding so far. The Ab I use is from Cell Signalling (#4771).
I am wondering whether there is anything I should be more cautious with when preparing lysates of acetylated proteins? (as for example phospoproteins are not stable so they need extra caution)
Any tips, greatly appreciated!
Try using different concentrations of the same antibody. You can make serial dilutions.
I hope this helps.
Hello Maria,
a smear of bands could be due to many things. For example: too much protein, too much detergent, the sample was insufficiently lysed, the gel concentration.
Multiple bands: how specific is the antibody for the protein you are looking for? Can your antibody detect other acetylated proteins too (could account for multiple bands of about the same signal intensity)? Is the band for your protein of interest stronger than the other bands? If yes, use less antibody. Can you increase the blocking time or the concentration of Tween in your blocking buffer to reduce unspecific bands?
Sorry for this many options, but good luck!
Gisela
hi
Before run the WB , Please read carefully the company data sheet >>
https://www.cellsignal.com/products/primary-antibodies/acetyl-cbp-lys1535-p300-lys1499-antibody/4771
"For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight."
you can do following :
1- before run the sample , determine the lysate concentration by BCA kit or ...
2- set up western blot with positive control by various concentration ( 20 ug/well ,40ug,60ug)
3- you can use from TSA-treated A431 or other cancer cell line TSA = Trichostatin A
4- you can use RIPA lysis buffer or modified RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% IGEPAL, 0.1% SDS, 0.5% Na deoxycholate)
5- Run the sample on 8% SDS-page , put runing tank and gel in ice tray or cold room (4c)
6- transfer mode = time 70 min / 100 v ( MW =300 kd)
7- incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight
good luck
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