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
- - - - -

Protein Size 50 kDa higher than expected


  • Please log in to reply
3 replies to this topic

#1 Tensix

Tensix

    member

  • Active Members
  • Pip
  • 9 posts
4
Neutral

Posted 07 September 2012 - 09:51 AM

We're having a curious problem with a western blot in our lab.

Our protein of interest is expected to be ~95 kDa, reported by multiple antibody providers, Ensembl, Uniprot, etc. Bizarrely, some of these antibody providers state the expected size is 95 right next to a gel image that shows it at 130. Others show a gel image with bands at both sizes. I've contacted 3 of them about this, 1 didn't answer and the other two took a week to admit they didn't have any explanation.

With two different antibodies in multiple blots, we see a strong band at ~150 and either nothing or a much weaker band at 95. We also have a commercial positive control lysate from a cell line transfected to overexpress the protein. In multiple blots with the two antibodies, it has bands at both sizes.

I'd chalk it up to cross-reactivity and assume the 95 is correct, except that I also did a transient siRNA knockdown. Comparing the target knockdown sample and the scramble knockdown sample, the 95 and 150 bands are present in both samples and both are markedly weaker in the target knockdown. Sample loading was even by B-actin. The siRNA knockdown was also tested by qPCR at the same timepoint and found to be very specific.

So that suggests both bands are in fact the target.

-There are multiple isoforms of the protein, but they are all nearly the same size (~95-100 kDa).

-None of the post-translational modifications I know of would account for a 55 kDa discrepancy except for possibly glycosylation- but there's no glycosylation of this protein on record, and both bands are tight on the blot with no sign of smearing.

My other thought is that there's a strong protein-protein interaction or some folding making it through the denaturation step, but I've tried pretty stringent conditions:

4x LDS NUPAGE loading buffer from Invitrogen + 0.45 ul B-mercaptoethanol ea sample
NuPAGE running and transfer buffers
4-12% Bis-tris 10 well gel
95º C boiling (5, 10, 15 minutes)

I'm kind of stumped at this point. What am I missing?

#2 ascacioc

ascacioc

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 271 posts
44
Excellent

Posted 07 September 2012 - 10:36 AM

some proteins crosslink like in the case of ubiquitin...maybe it is an interaction partner that is covalently linked? This would stay together at 150 kDa. I would make it somehow that I have the band on an SDS-PAGE gel and send it for mass spec.

#3 proteaMatt

proteaMatt

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
4
Neutral

Posted 10 September 2012 - 04:54 AM

If you are going to send it somewhere to have it analyzed by MS it would be helpful to see how your protein of interest looks on a 2D gel. You could even blot it, which might help give you a better picture of what is going on.
Lab Technician at Protea Biosciences

#4 Missle

Missle

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 132 posts
18
Good

Posted 10 September 2012 - 11:40 AM

Sometimes proteins that are extremely charged can run completely different than expected on SDS-PAGE. My lab is currently working with one such protein whose pI is ~4.2 and is only ~32kD yet consistently (and is commonly known in the literature to) runs at ~60kD by SDS-PAGE. To make things more confusing, any degredation or cleavage products will also run oddly, making it extremely difficult to determine size by this method. The gel images provided by the antibody companies support this being a characteristic of your protein. The following link explains some of the other reasons for abnormal migration in SDS-PAGE (proline content, charge, etc) and gives examples.
http://www.bio-rad.c...lletin_3133.pdf

Good luck!




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

©1999-2013 Protocol Online, All rights reserved.