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
- - - - -

Cloned sequence is not predicted size by western blot

Cloning Western blot Kilodalton Predicted size

  • Please log in to reply
1 reply to this topic

#1 Rara

Rara

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 31 January 2012 - 03:03 PM

Hi, we sub cloned a DNA sequence into our vector of choice. The sequence subcloned was predicted to give a protein molecular weight of 36 kda. When western blot was performed of transduced cells ( expressing the gene of interest), we got a very good western blot signal. However the molecular weight of the protein detected was 48 kda (using a nuMber of different antibodies). Does anyone have an idea of why our cloning is generating a protein that is 10kda heavier than that is predicted by its DNA sequence. It seems unlikely to be phosphorylation etc.. We believe we have completely denatured the protein lunate, including further 'linearisation' using urea.
Cheers
Alex

#2 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
5
Neutral

Posted 01 February 2012 - 12:44 AM

posttranslational modifications, not necessarily phosphorylation but could also be glycosylation or any other, seem to be the simplest explanation.





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

©1999-2013 Protocol Online, All rights reserved.