High background on westerns - (Feb/28/2008 )

HI All,
I used the following protocol for immunodetection by G:Box (CCD) system.

1 hr blocking in 5% (nonfat dried milk) in TBST for 1 hour, primary antibody incubation at 4 degree c overnight (1: 500 dilution)in TBST +1%BSA, followed by washing and 1 hour incubation at room temperature with secondary antibody (1:10,000 diln, in TBST) and washing, ECL and subjecting it to G: box, I encountered high background, could anyone please suggest me how I can reduce the high background. I used Immuno-PVDF (Bio-rad).
I appreciate your help.

At the beginning, I wash with TBST 3 times, 10 minutes each time. There's background so I later wash the membrane with TBST 6 times, 5 minutes each time. I found that the background has been reduced!

Also, use 1%-2% non-fat milk with your secondary anitbody, this will also reduce the background.


i don't know is it co-incident or I just simply improved my skills, but my background improved alot when I used PBST 6 times 5 mins each after primary antibody --> use TBST 6 times 5 mins each after secondary antibody incubation. Since i have no background ever since, i stick with this method.. haha...


Hope this helps!

like tulip said, try to dilute secondary and/or primary Ab in non-fat milk. I use up to 5% with both AB and Blocking. In my hands BSA does not give nice blots.

Thanks for the inputs. I will try. Has anyone tried using SEABLOCK from Pierce?

Hi, normally high background is caused by inefficient blocking. There are some antibodies that work well in BSA, others in milk. So, if your antibody should work fine in 1% BSA (as described by manufacturer instuctions, or other), try to block in 3% BSA.

Milk blocking is more efficient than BSA blocking, so get BSA's blocking step for more time, try overnight for instance..

I used some similar blocking solution from Pierce, but i didn't notice improvements in my conditions, of course. Anyway, my suggestion is take more time in blocking step, and use same solution for primary and secondary antibodies (i.e. block in milk/Ab in milk; block in BSA/Ab in BSA)

An other thing could be Tween concentration. I normally use 0.1% final.

let me know is if it's better..

bye!
S.75