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
- - - - -

Increase in protein size


  • Please log in to reply
5 replies to this topic

#1 eukaryote

eukaryote

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 17 October 2009 - 09:37 AM

Dear Friends, Please help me solve this strange problem quickly
I used Pet 30 a vector and Rosetta 2 de3 cells. I expressed 2 proteins, and one of them should be 22 kda but it is showing 29 kda and another one should be 17 but its like 23 kda on SDS-PAGE. I added his tag and other tags inframe when expressed. When I used nickel column, the protein bound well and all the fractions showed the same increased size and eluted well. Has any one experienced this problem. If you please let me know how you solved your problem, that woud be very helpful. I look forward to hear your words of wisdom as soon as possible.
Thank you

#2 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 18 October 2009 - 03:33 PM

Dear Friends, Please help me solve this strange problem quickly
I used Pet 30 a vector and Rosetta 2 de3 cells. I expressed 2 proteins, and one of them should be 22 kda but it is showing 29 kda and another one should be 17 but its like 23 kda on SDS-PAGE. I added his tag and other tags inframe when expressed. When I used nickel column, the protein bound well and all the fractions showed the same increased size and eluted well. Has any one experienced this problem. If you please let me know how you solved your problem, that woud be very helpful. I look forward to hear your words of wisdom as soon as possible.
Thank you

What RE site(s) did you use? Could you have removed a stop codon?
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#3 lna

lna

    member

  • Active Members
  • Pip
  • 26 posts
1
Neutral

Posted 22 October 2009 - 05:00 AM

Did you always have the increased size of the products with this vector, or it occured after some manipulations? Because sometimes you can get the translation from the alternative start-codon, which is not even an AUG.. in this case you can only try to make the shift in the reading frame, which corresponds to the amternative initiation point. Of course, if you have the His-tag in the N-terminus, it could not be the case. In This case there could be a loss of stop-codon. So, check the sequence?

#4 swanny

swanny

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 367 posts
8
Neutral

Posted 25 October 2009 - 05:41 PM

Also, is there anything strange about the amino acid sequence? Sometimes proteins will run at a different apparent mass.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#5 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 25 October 2009 - 08:08 PM

Also, is there anything strange about the amino acid sequence? Sometimes proteins will run at a different apparent mass.


Also post transationally modified proteins like glycosylated ones show a higher molecular weight in gels due its hydrodynamic radius!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#6 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 25 October 2009 - 08:12 PM

Also check for the mol wt any any other orthogonal technique just o be sure!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!




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

©1999-2013 Protocol Online, All rights reserved.