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
- - - - -

His-tag protein vs. BSA


  • Please log in to reply
3 replies to this topic

#1 Yasser Almeida Hernández

Yasser Almeida Hernández

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 28 April 2011 - 10:30 AM

Hi all...
I'm purifying a 105 kDa glycoprotein from mammalian cell culture using Cu. As know, the mammalian cell culture often require a percent of fetal bovine serum wish contain huge amounts of bovine serum albumin (BSA). I'm using imidazole at 2, 10 and 100 mM in the elution buffer and in the all cases my protein and the albumin co-eluted with my protein as well as other minority contaminant proteins. I know that the Ni (Ni-NTA) in the most commonly used ion i this type of purification but I don't have the Ni-NTA column neither the salt itself right now.

The BSA have 17 histidine along all the sequence, but, as the quantity is so high, the protein stick to the column, i think...

So, do you have any suggestion to eliminate the albumin in the purification? That is normal?

Thanks all in advance...

#2 proteaMatt

proteaMatt

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 63 posts
4
Neutral

Posted 28 April 2011 - 01:58 PM

What is the volume of your sample that you want to purify? Do you know the approximate mass of the BSA in your sample?

An albumin depletion kit can deplete 90% or more of the albumin from your sample so long as it falls within the parameters of usage. Usually, I recommend samples of <100 µL that contain up to 500 µg of albumin. I have found it to work really well on a 10% fetal bovine serum growth media, so my recommendation would be to harvest your cells as normal and then deplete the albumin from your sample before processing it with your Cu. The other thing to keep in mind is that following albumin depletion the sample will be buffered at a pH of around 7 with a phosphate buffer. So make sure that wouldn't interfere with your following steps.

Edited by proteaMatt, 28 April 2011 - 02:01 PM.

Lab Technician at Protea Biosciences

#3 protolder

protolder

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 293 posts
9
Neutral

Posted 28 April 2011 - 10:13 PM

Hola, in all the methods low concentration of imidazole about 10-20 mM are added to bound buffer and wash buffer in order to eliminate the inespecific interactions, washing this inespecific bounded protein and remaining bound 6His tagged proteins wich eluted with high imidazole concentrations. Your case is extrange could be a specific interaction of your protein with BSA? because the affinity of BSA and the 6HIS tagged protein have to be different, or the pH of your buffer is too low? avoiding the bound . Buena suerte

#4 Yasser Almeida Hernández

Yasser Almeida Hernández

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 29 April 2011 - 07:49 AM

THANKS ALL... :)




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

©1999-2013 Protocol Online, All rights reserved.