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 purification problems: Presence of contaminated proteins along with the


  • Please log in to reply
5 replies to this topic

#1 Bioscientist

Bioscientist

    member

  • Active Members
  • Pip
  • 26 posts
1
Neutral

Posted 16 January 2013 - 10:06 AM

Hi all

My protein is His-tagged and has a molecular weight of 27 KDa. I expressed it in Rosetta E.Coli cell. I used a 1 litre culture to extract soluble protein. After purification using Ni-NTA column I ended up with multiple bands along with the protein of interest.

IMAC purification was done as follows:

The colum was equilibrated with 2 column volumes (20 ml)
Equilibration buffer: 50mM Tris-HCl, 100mM NaCl, pH 8.0.

Then the protein with Ni-NTA agarose flowed through the column.

Then washed with wash buffer 1 (50mM Tris-HCl, 100mM NaCl, 20mM imidazole pH 8.0) twice.

wash buffer 2 (50mM Tris-HCl, 100mM NaCl, 40mM imidazole pH 8.0) once.

Elute buffer 1 (50mM Tris-HCl, 100mM NaCl, 100mM imidazole pH 8.0) once 1ml.

Elute buffer 2 (50mM Tris-HCl, 100mM NaCl, 250mM imidazole pH 8.0) 4 times 1 ml each.


I understand number of washing steps and imidazole concentration helps to remove contaminated proteins. But in the washing steps there is almost no proteins. 100 mM has some in the elution 1. and in elution 2 there are multiple bands. So 250 mM works for both my proteins and the contaminated proteins.

I have attached a picture of my gel run. I use DTT in the loading buffer during running SDS-PAGE gel. From L-R; flow-trough, wash1-1st flow, wash1-2nd flow, wash 2, elute 1, elute2-1st flow, elute2-2nd flow, elute2-3rd flow, elute2-4th flow, ladder.

I appreciate any help in this matter.

Bioscientist.

Edited by Bioscientist, 16 January 2013 - 10:08 AM.


#2 HOYAJM

HOYAJM

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 159 posts
12
Good

Posted 16 January 2013 - 11:01 AM

In my opinion, the elute2-2nd flow looks pretty good. From my experiences, the lower band you are seeing might be very difficult to remove and is somewhat ordinary. You do seem to have a conundrum since 100mM is starting to elute your protein of interest but not all of the contaminants. I recommend changing elute 1 to 125-150mM imidazole and think of it as more of a wash. Then elute with 300mM imidazole and see if the first flow through is cleaner.

The bands you have though are very nice and when I was reading your post it made it sound like you had TONS of contaminant bands, but in reality those are pretty clean fractions. If it is available to you, size-exclusion chromatography would clean these samples up very easily.

#3 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,437 posts
241
Excellent

Posted 16 January 2013 - 04:51 PM

I believe you should declare success and move on. There is a little contamination, which is very typical for His tagged protein purification, but your desired product is way more prevalent. Almost certainly this is pure enough for most purposes.

#4 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 17 January 2013 - 07:29 AM

Agree with HOYAJM and phage434. I myself rarely got such a clear bands like yours.
As long as your background protein doesn't interferes with your downstream work, it should be fine.
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 Bioscientist

Bioscientist

    member

  • Active Members
  • Pip
  • 26 posts
1
Neutral

Posted 18 January 2013 - 01:48 PM

Thanks a lot for the replies. I was wondering if I use size exclusion chromatography what media or resin should I use for the chromatography?

#6 Adrian K

Adrian K

    Legendary Graduate Beggar

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 708 posts
28
Excellent

Posted 20 January 2013 - 08:49 AM

Example, you can refer to:
https://www.gelifesc...20420130859.pdf

Considering your protein is quite pure and the contaminant is not close to your protein of interest, You can consider to use HiPrep 16/60 Sephacryl S-100 HR, which is relatively cheaper than superdex ranges.
However, you need to ensure there is a proper FPLC or HPLC for you to use.

You can also consider other brands of column.
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




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

©1999-2013 Protocol Online, All rights reserved.