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
- - - - -

Factors interfereing with chemiluminescence


  • Please log in to reply
3 replies to this topic

#1 krithbala

krithbala

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 29 March 2011 - 08:29 PM

What interferes with chemiluminescence? We transferred 100 ug equivalent of protein (from lysate) on to nitrocellulose using semi-dry transfer. Ponceau staining of the blot showed that the transfer had occurred with all samples (efficiency is still a question). When probed with primary and secondary, the chemiluminescence worked only with the standards and one of the lysates. What could be the problem? All samples have the antigen.

#2 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 29 March 2011 - 10:13 PM

What interferes with chemiluminescence? We transferred 100 ug equivalent of protein (from lysate) on to nitrocellulose using semi-dry transfer. Ponceau staining of the blot showed that the transfer had occurred with all samples (efficiency is still a question). When probed with primary and secondary, the chemiluminescence worked only with the standards and one of the lysates. What could be the problem? All samples have the antigen.

Hola I think that your chemoluminiscent substrate runs, because all the antigen samples are in the same membrane, or not?. Depending of the epitope against the ab is made or if it is made against well folded or denatured whole protein it will expose or nor the epitopes to the ab and it will reaction or not. Check how ab was made and try to see the explanation or return here for give us more details. Buena suerte

#3 almost a doctor

almost a doctor

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 244 posts
15
Good

Posted 30 March 2011 - 12:29 AM

What interferes with chemiluminescence? We transferred 100 ug equivalent of protein (from lysate) on to nitrocellulose using semi-dry transfer. Ponceau staining of the blot showed that the transfer had occurred with all samples (efficiency is still a question). When probed with primary and secondary, the chemiluminescence worked only with the standards and one of the lysates. What could be the problem? All samples have the antigen.


Could you give us a bit more detail of what you've done.

What are your standards? Is this a positive control? As for the lysates, have these samples all been run in the same gel, same membrane, and therefore incubated together? Or is this different gels and/or membranes incubated separately? What antibody concentrations are you using? How long do you incubate for? How do you block and wash?...

One of the problems could be your secondary antibody concentration being too high, as this could exhaust the ECL substrate even before you have time to measure it. Which brings me to my final question, how are you performing your detection: Film, CCD camera?

We need your help to help you, the more info you can give us the better ;)

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,830 posts
415
Excellent

Posted 03 April 2011 - 03:49 PM

Are your samples degraded? how have they been stored? do you add protease inhibitors?




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

©1999-2013 Protocol Online, All rights reserved.