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
- - - - -

Huge background with HT22 cells phophoERK antibody

Western blotting he western blotting

  • Please log in to reply
3 replies to this topic

#1 sikander

sikander

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 12 December 2011 - 05:54 AM

Hi all,

I am studying the ERK activation in HT22 cells and for this first i probe my blot with total ERK and got good signal and later on when i do stripping. After stripping the blot i prob the blot with phosphoERK antibody.

My total ERK gives very good signal, while there comes huge background in the phospho ERK probing.

Can anyone gives some suggestion.

thanks

#2 GNANA

GNANA

    GNANA

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 209 posts
9
Neutral

Posted 13 December 2011 - 04:07 AM

Always do the Phospho's first and then do the total....Phosphorylations would get affect on stripping...background might be due to various factors including the antibody dilutant...sometimes if there is no detectable phosphorylation on blot would give backgrounds....hope your lysis buffer has the phosphatase inhibitors, have a check at this also...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 acquire

acquire

    member

  • Active Members
  • Pip
  • 9 posts
0
Neutral

Posted 28 December 2011 - 04:18 PM

phospho ERKs generally i keep for overnight at 4centigrade,then it works good.myself uses 1:1000 times dilution primary
and 50ug protein/well.and ofcourse its always good detect phospho ERKs first or else use 2 different gels or blotts for ERK ans phosph ERK.

#4 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
135
Excellent

Posted 29 December 2011 - 02:36 PM

with what do you block (and add to your antibody solution)?

if you use milk then that could be the cause of your background. milk contains phosphoproteins (e.g. casein).
talent does what it can
genius does what it must
i do what i get paid to do





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

©1999-2013 Protocol Online, All rights reserved.