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
- - - - -

Problem with strep tagged protein purification

strep tag purification

  • Please log in to reply
5 replies to this topic

#1 origami

origami

    member

  • Active Members
  • Pip
  • 5 posts
1
Neutral

Posted 06 August 2013 - 07:53 AM

Hi,

 

i am working with a N-strep tagged, cleavable by 3C, hydrophilic protein and i see it on the FT and after regenerating the column (GE healthcare, 5 ml) with 0.5M NaOH i see again the protein.

 

i thought that:

-either the affinity (material-protein) is too strong,

-or my protein is aggregated (i did SLS with the eluted protein after the regenaration and the peak and MW corresponds to the protein, therefore i excluded this thought),

-or the concentration of the loaded protein is too high (the lysis buffer was 1/100 and i filtered before loading it onto the column, therefore i excluded this thought),

-or the flow rate is not the proper (i tried with 3ml/min and 5ml/min, the result was the same, therefore i excluded this thought)

-or the strep tag is hidden (i add to the lysis buffer up to 10mM b-merca, therefore i excluded this thought)

-or the d-besthiotin was not enough (i add up to 10mM d-besthiotin, therefore i excluded this thought)

-or the pH of the buffer was below 7 (i checked the buffer and it was fine)

-or to increase the NaCl (up to 250mM, again the same result).

 

 

my last hope is to do the tag cleavage on the column overnight and hopefully the next day to get my protein in the flow through. Additionally, to add up to 1% Triton X100 to the lysis buffer.

 

any suggestion is wellcome!

 

 

 

 



#2 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 11 August 2013 - 08:56 AM

Are you using syringe or chromatography system to clean your column?

How many column volume of buffer you use to regenerate your column?

Can you show us your chromatography profile?


Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#3 origami

origami

    member

  • Active Members
  • Pip
  • 5 posts
1
Neutral

Posted 11 August 2013 - 11:20 AM

i am using FPLC and i do 3-4 CV regenaration.

 

 

finally, i solved my problem with the on column tag cleavage. i guess the protein interacts very high with the material of the column and finally after the tag cleavage i get the protein.

 

i tried also using peristaltic pump and i had the same results.

 

thank you very much



#4 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 707 posts
28
Excellent

Posted 12 August 2013 - 09:33 AM

Glad to hear that. Congrats.

Just curious, based on my understanding that we need to do at least 5 CV for washing and regeneration. Any reasons to share on using lesser CV as you did?

 

:)


Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#5 origami

origami

    member

  • Active Members
  • Pip
  • 5 posts
1
Neutral

Posted 12 August 2013 - 12:30 PM

yea, i thought the same and i used to do before like this but i didnt see any difference between 3-4 CV to 5-6 CV.



#6 littlebull

littlebull

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 19 August 2013 - 11:49 AM

Your protein either does not binds to the column (e.g losing tag) at all, or weakly binds to the column.

 

1. no binding: most of your protein is in flow through, the rest aggegates on column which was washed away by 0.5 NaCl.

 

2. weak binding: you can load your protein twice, or add resin to your protein solution and incubate for a few hours or even overnight.

 

Good luck






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

©1999-2013 Protocol Online, All rights reserved.