Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

High background....


  • Please log in to reply
6 replies to this topic

#1 Danny Chow

Danny Chow

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 12 May 2009 - 09:05 AM

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

#2 Aris

Aris

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 67 posts
0
Neutral

Posted 12 May 2009 - 04:56 PM

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



What kind of II Ab are you using? is it anti-goat?
Try raising the milk from 3% to 5% all the way through

#3 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,728 posts
399
Excellent

Posted 12 May 2009 - 05:56 PM

titrate your secondary out, you probably need to dilute it more.

#4 medchemgirl

medchemgirl

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 69 posts
1
Neutral

Posted 20 May 2009 - 12:57 PM

You can also try incubating your antibodies with PBS-T. And do a few washes with PBS-T then with PBS

#5 Irene G

Irene G

    member

  • Active Members
  • Pip
  • 17 posts
0
Neutral

Posted 21 May 2009 - 06:33 AM

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

#6 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 21 May 2009 - 07:55 AM

5) rinse the membrance with 0.5% tween 20 for 15mins


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.

#7 rajgene

rajgene

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 34 posts
1
Neutral

Posted 01 July 2009 - 06:21 AM

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.

Edited by rajgene, 01 July 2009 - 06:24 AM.





Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.