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!
- - - - -

Need to strip the membran before probe the internal control protein

  • Please log in to reply
3 replies to this topic

#1 CarrieX



  • Active Members
  • Pip
  • 5 posts

Posted 31 December 2013 - 07:08 AM

Hello! I am new in the proteomic area. I need to do a quantitative western blot and use Actin as internal control. I am not sure how to blot probe two proteins in one membrane (my target protein and Actin). Can I  cut out the area for Actin (~50kDa) and probe anti-Actin antibody and probe the other area with my target antibody? Or do I need to first probe my target protein and strip and reprobe the Actin? 





  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 220 posts

Posted 31 December 2013 - 07:24 AM

If the target protein and actin are far enough to be able to cut and probe, that would always be my choice instead of striping.

I always had an alternate hypothesis....

#3 jerryshelly1



  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts

Posted 31 December 2013 - 09:23 AM

I am always nervous cutting blots and actin antibodies show such low cross-reactivity that I always order my experimental proteins with different conjugations to my internal control. This allows me to blot for both proteins simultaneously and if you are using a fluorescent secondary, it makes a beautiful blot. Maybe consider using a biotinylated antibody control in the future so you know there is almost zero crossreactivity. 

#4 bob1


    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 6,674 posts

Posted 03 January 2014 - 02:28 PM

I agree with jerryshelly1 - so long as your two proteins are sufficiently separated, probe for the actin without stripping or cutting the blot.  I do this as standard using normal HRP based secondaries and x-ray film and very rarely have problems separating 50 kDa proteins from actin at 46 kDa.  It helps if you can use a ladder which shows up when probed, such as MagicMark from Invitrogen, but it isn't essential.

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

©1999-2013 Protocol Online, All rights reserved.