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
- - - - -

YFP staining


  • Please log in to reply
2 replies to this topic

#1 scistudent

scistudent

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 22 May 2009 - 04:47 AM

Hi, I am having problems with my YFP staining. I am transfecting cells with YFP (and see it during the time in vitro, so it's def there). But when I stain for it, I just get lots of back ground and very very few cells stained... anyone had this problem too? Its a santa cruz antibody and I am fixing with methanol and PFA. I have tried increasing the ab concentration and am going to try blocking at a higher % too.

Any ideas would be great, thanks!

#2 Carlton H

Carlton H

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 95 posts
0
Neutral

Posted 24 May 2009 - 02:05 PM

A few things...

1) If you have too much background, that generally means that you have too much antibody, and are therefore getting nonspecific staining. Try reducing the concentration of antibody and see if that reduces your background.

2) lots of background could be a result of your fixing. If you fix for too long, that could be one possible source of the high background. How long are you fixing for? ... Also, you could try removing the methanol from the fix procedure, as dehydration may (relatively rarely) change the conformation of the protein enough such that the antibody will no longer recognize it.

3) I hate to say anything negative about a company, but Santa Cruz antibodies have a reputation for being less than wonderful. Be sure to preabsorb your antibody (ideally you would use non-transfected cells for preaborption, since these will have all the same proteins except no YFP). If you can borrow another antibody from someone else (or buy one), then try that as well. It's totally possible that the antibody may just be no good. Do you have any confirmation that it even works for IHC?

If all else fails, you could always transfect with a different FP (GFP, etc.) and see if that works.

Let me know if you still have problems.

Cheers,
-Carlton
NextAdvance
Reliable laboratory instruments for the life sciences, designed to enable you to work more effectively and increase productivity. Check us out!

If my answers help you, please take a moment to check out our site and see if any of our products would help you as well! Thanks!

#3 scistudent

scistudent

    member

  • Active Members
  • Pip
  • 26 posts
0
Neutral

Posted 09 June 2009 - 06:50 AM

Hi,

Thanks very much for that! I havn't heard great things about Santa Cruz either but I couldn't find another YFP ab... also what do you mean by pre-absorb the antibody? I fix for 10 minutes with methanol and then 15 mins with PFA 4%, so I wil try leaving out the methanol this time and use less ab and see how it goes.

Thanks again!




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

©1999-2013 Protocol Online, All rights reserved.