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
- - - - -

AnionExchange problem: proteins do not bind


  • Please log in to reply
12 replies to this topic

#1 mirc

mirc

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 27 May 2010 - 03:25 AM

Hi folks,
Iīm an absolute beginner with regard to protein purification, but want to do some traditional protein clean-up (Anion/Cation EX; Gelfiltration).
Iīm having tremendous problems with the first step: an anionexchange column (1ml Q XL from GE).
Whenever I load a cell lysate sample (~20mg, 2ml sample volume) nearly everything is in the flow through fraction and nothing really binds to the column as judged from WB and the chromatogram (I run: 1. 6ml 50mM NaCl buffer, then 15ml with a gradient form 50-1000mM NaCl; flow 0,5ml/min).
I donīt want to go below 50mM NaCl, cause Iīm actually interested in a complex my protein of interest forms. But regardless of the 50mM more proteins should bind to the column initially...
Before adding the lysate on the column, the lysis buffer (150mM NaCl, 0,25% Deoxycholate, 1%NP40, etc, no SDS ) is exchanged to start buffer with a desalting column ( also tried dialysis).
Iīve also tried Glycerol in the buffers with the same result.

Can anyone give me a hint how to get my proteins to bind???

Thank you very much in advance!

post also posted in "Biochemistry"

Edited by mirc, 27 May 2010 - 03:52 AM.


#2 Inmost sun

Inmost sun

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 326 posts
60
Excellent

Posted 27 May 2010 - 06:40 AM

you may think of starting with hydrophobic interaction chromatography where you can start with high salt concentartions; my experience is that Q-columns are better at a late step when you have gotten rid off of bulk proteins...

which buffer and pH do you use?

#3 paramyosin

paramyosin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
0
Neutral

Posted 27 May 2010 - 08:00 AM

Something is wrong, the mayority of the E. coli proteins (i assume is E. coli if not donīt read the following) are acidic, therefore they should bind to the column at pH of 7 (which pH you use in your buffer?). I think you are using too much extract just for 1 ml column, why donīt you use 200-500 ul of seed?. Are you analyzing your elution fractions by a SDS-PAGE? if you are controling just your FT probably you wouldnīt see nothing because you have too much protein. What do you use for your WB?, a monoclonal Ab against the protein or against a tag?. Maybe you should check also in a SDS-PAGE to see that you have protein because you donīt know if your protein is not binding to the resin or if the resin is not working with any protein.
Are you sure that you are using the correct pH regarding the pI of your protein in order to assure that it is binding to the resin?
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#4 mirc

mirc

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 27 May 2010 - 08:05 AM

>which buffer and pH do you use?

2mM Tris pH8
1mM MgCl2
50-1000mM NaCl

Unfortunately we donīt have a hydrophobic interaction column but maybe I should start off with a gel filtration, though that would be an uncommon first step...

Itīs U2OS cell lysate, not e. coli, but at ph8 there should be a lot of negatively charged protein. Calculated pI of the protein is ~5, so should be negative. The dynamic binding capacity is stated as >130mg BSA/ ml bed volume, so I shouldnīt have overloaded the column, or do I misunderstood something? Also the volume shouldnīt matter with IEX....Iīm using a polyclonal AB against the protein btw....

Edited by mirc, 27 May 2010 - 08:15 AM.


#5 Inmost sun

Inmost sun

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 326 posts
60
Excellent

Posted 27 May 2010 - 01:00 PM

>which buffer and pH do you use?

2mM Tris pH8
1mM MgCl2
50-1000mM NaCl

Unfortunately we donīt have a hydrophobic interaction column but maybe I should start off with a gel filtration, though that would be an uncommon first step...

Itīs U2OS cell lysate, not e. coli, but at ph8 there should be a lot of negatively charged protein. Calculated pI of the protein is ~5, so should be negative. The dynamic binding capacity is stated as >130mg BSA/ ml bed volume, so I shouldnīt have overloaded the column, or do I misunderstood something? Also the volume shouldnīt matter with IEX....Iīm using a polyclonal AB against the protein btw....


you can combine cationic and anionic exchanger chromatography; I routinely started with cationic exchanger chromatography, then hydrophobic interaction chromatography then Q column...

#6 paramyosin

paramyosin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
0
Neutral

Posted 28 May 2010 - 07:00 AM

The dynamic binding capacity is obtained after several hours of interaction between the resin and a substrate, the real binding capacity depends on the residence time. Definitely I would try with less initial volumen, i think that you are using too much for your 1 ml column. The sample load is an important parameter in resolution. Are you using FPLC? with very high loads, sample displacement can occur. I agree about cation and anion exchange, sometimes you can have surprises regarding the real pI of your target protein.
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#7 mdfenko

mdfenko

    an elder emeritus

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 3,292 posts
202
Excellent

Posted 28 May 2010 - 11:19 AM

you might want to consider increasing the concentration of tris to at least 20mM. 2mM won't do a very good job of maintaining the pH.
talent does what it can
genius does what it must
i do what i get paid to do

#8 Prep!

Prep!

    Am I me???!!!

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 517 posts
4
Neutral

Posted 29 May 2010 - 02:55 AM

conductivity and pH are the two factors which will decide the binding on Q column.. i don know abt bacterial products but with cell culture based products Q is a very very common first step!!!!
Support bacteria - They are the only culture some people have!!!
Cheers!!!

#9 mirc

mirc

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 30 May 2010 - 10:05 AM

@paramyosin:
Sorry, that was typo; I do use 20mM Tris and an FPLC System
What do you mean by "sample displacement can occur"?
I will try with a 500ĩl loop though. I just thought that sample volume wouldnīt matter with IEX.

#10 paramyosin

paramyosin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
0
Neutral

Posted 31 May 2010 - 06:27 AM

the sample displacement is a phenomenon that can be occur at very high loads due to overlap between solute zones which may reduce yield and/or purity of your target solute. The overload effects may be avoided by restricting the load..... However you say that you donīt have protein at all binded to the resin, have you?. Are you meassuring your conductivity?, i woul try with a initial one under 10 mS/cm and afterward you can eliminate the protein in which you are not interested in (maybe 50 mM is too much and you can loose your protein), try just to be sure that you are not loosing your target protein. Then you say that you perform a 15 CV gradiente between 50 and 1 M NaCl. I havenīt got a clear idea about your chromatogram, have you got peaks in it?, i am talking about other proteinsīpeaks in order to know if your column is properly working. Can you send the file of the chromatogram?
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#11 mirc

mirc

    member

  • Active Members
  • Pip
  • 5 posts
0
Neutral

Posted 01 June 2010 - 03:40 AM

sorry for responding so late,
The column does bind some protein (only a little in comparison with the flow through peak (~aprox1%). If I scout for another protein via WB it seems nicely bound, but my protein of interest isnīt. The problem is, that it was retained once (my initial experiment) but ever since my protein of interest doesnīt bind any more...
Unfortunately I canīt measure conductivity...

#12 paramyosin

paramyosin

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
0
Neutral

Posted 09 June 2010 - 01:30 AM

Different proteins bind differently in a resin!, but if you have good binding of other protein is because the resin is ok. Maybe your initial conditions were different, more NaCl concentration?, different pH?, different resin-protein relation?
"Research without indebtedness is suspect, and somebody must always, somehow, be thanked." Umberto Eco

#13 NHI

NHI

    member

  • Active Members
  • Pip
  • 7 posts
0
Neutral

Posted 12 July 2010 - 11:14 PM

hi fren...

by the way u have to see the principle of Ion exchange chromatography...let me explain..

since u use Q resin....i do believe it is positively change resin.if u want to make ur protein bind to the resin, u should make ur protein as -vely charged.HOW?????

Do u know the isoelectric point of your protein????if not, u should make an isoelectric focussing (IEF) experiment...then, from the pI value that u will get, u can know what is the appropriate pH for ur sample to ensure the it will bind...

i give u example, let say the sample is IgG....u spin down ur sample and run IEF in order to get the pI value...
let say the pI is between 7.6 to 8.2....by assuming an electrophoretic titration curve, i.e. lower the value from 7.6 will make the protein as +vely charge, whereas increase the value from 8.2 will make the protein as -vely charge...

let say u want the IgG bind to Q resin, so u must choose pH above 8.2...let say u set pH at 8.6

Then, u choose any buffer that has buffering region at this chosen pH....at pH 8.6, Buffer Tris-HCl (10 mM) can help...then, u dialyze/ buffer exchange ur sample to this buffer...

Secondly, by rule of thumb....for ion exchange chromatography binding mode (i.e. product of interest will bind to resin), the conductivity of sample must less than 5 mS/cm...so, use buffer at low strength...such as 10mM or 20 mM....


by the way, good luck...




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

©1999-2013 Protocol Online, All rights reserved.