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
- - - - -

Bands are lower than actual protein


  • Please log in to reply
1 reply to this topic

#1 Aravin

Aravin

    member

  • Active Members
  • Pip
  • 14 posts
1
Neutral

Posted 31 August 2010 - 07:29 AM

Hello,

I'm doing a Western to quantify the activity of P21-activated kinase (PAK) by using a phospho-serine/threonine antibody. I first pulled-down alpha-PAK with an antibody and then added Protein G Agarose beads. I then run the samples and total cell lysates on a 10% gel and immunoblot with the pSer/Thr antibody for the samples and alpha-PAK antibody for total cell lysates (as a loading control). My problem is that my pSer/Thr bands do not match up with the total PAK bands. PAK is 65 kDa and is on the right side of the blot I have attached. Does anyone have any suggestions or ideas of what the problem may be?

Thanks

Attached Thumbnails

  • blot1.jpg


#2 HomeBrew

HomeBrew

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 930 posts
16
Good

Posted 31 August 2010 - 06:32 PM

Multiple epitopes to one antibody and not the other? Quantitative westerns are a tricky proposition...




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

©1999-2013 Protocol Online, All rights reserved.