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
- - - - -

GAPDH merging lanes


  • Please log in to reply
1 reply to this topic

#1 Nezura

Nezura

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 10 February 2011 - 07:42 PM

Hi there I'm getting bands like this for my GAPDH control and I'm not sure why the bands aren't more defined.

I'm running a 5% stacking and 10% separating gel. Gel is run at 100V for about 2 hours. Transfered to PVDF membrane at 100V overnight wet with ice block (can this be it?). Using a HRP staining system

Another problem I'm having is on the same gel but probed with a different antibody. The protein I'm interested in should just be one band at 76kDa (bottom visible horizontal lane of second gel) but I'm getting a whole bunch of staining on the top. I never used to get this. Could it be because I'm using a new lot of the same antibody from the same manufacturer? Is it non-specific staining or is my protein just getting separately improperly?

Thanks!

Attached Thumbnails

  • gapdh + ladder.jpg
  • protein of interest.jpg

Edited by Nezura, 10 February 2011 - 07:44 PM.


#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,811 posts
136
Excellent

Posted 11 February 2011 - 12:43 PM

for the second question, it looks like the antibody is not as specific as it should be.

for the first question, what do you mean by not well defined? i see them running together, a little. that can happen for a variety of reasons such as: letting the gel sit after running, allowing the proteins to diffuse; equilibrating with transfer buffer a little too long; a little too much protein loaded; a little too much salt in the sample; other additives in the sample; etc.
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.