High background.... - (May/12/2009 )
My protocol,
1) 5% skim milk in PBS blocking at RT for 1 hr
2) rinse with PBS, diluting primary anti with 3% BSA in PBS
3) Wash the membrane with 0.5% Tween 20 (20min/wash, total 3 washes)
4) 2nd antibody , diluting with 3% BSA in PBS
5) rinse the membrance with 0.5% tween 20 for 15mins
i got a very high background, the max time for me to expose the film on the membrane after applying ECL is 3s
If more than 3s, the whole thing will become black
Anyone got any idea so that i can have a clear background?
I try diluting more with the primary antibody, but the background didnt reduce....just the sensitivity reduced...
Thanks
Danny Chow on May 12 2009, 10:05 AM said:
1) 5% skim milk in PBS blocking at RT for 1 hr
2) rinse with PBS, diluting primary anti with 3% BSA in PBS
3) Wash the membrane with 0.5% Tween 20 (20min/wash, total 3 washes)
4) 2nd antibody , diluting with 3% BSA in PBS
5) rinse the membrance with 0.5% tween 20 for 15mins
i got a very high background, the max time for me to expose the film on the membrane after applying ECL is 3s
If more than 3s, the whole thing will become black
Anyone got any idea so that i can have a clear background?
I try diluting more with the primary antibody, but the background didnt reduce....just the sensitivity reduced...
Thanks
What kind of II Ab are you using? is it anti-goat?
Try raising the milk from 3% to 5% all the way through
titrate your secondary out, you probably need to dilute it more.
You can also try incubating your antibodies with PBS-T. And do a few washes with PBS-T then with PBS
Try to block with BSA 5% instead milk, with some antibodies it can works. And usually we used TBST rather than PBS but I don't think this could be the problem.
And 2 hours of blocking better.
Irene
Danny Chow on May 12 2009, 06:05 PM said:
Are you adding your ECL straight after step 5? You need to wash your membrane with just PBS (or TBS) without detergent before ECL incubation. I cant remember why but ECL will react with tween increasing your background.
Also, I'll recomend more washes for shorter times, the more times you change your wash buffer, the cleaner your membrane will be.
the problem here is with your secondary antibody. wash membranes three times the same way you do to for the primary antibody. high background is simply becoz of unwashed secondary antibody. if using a goat secondary antibody, you have to wash atleast 5 times as these goat primary and secondary are very sticky in nature.
i simply wash in PBS thrice for mouse rabbit antibodies. its works perfect.