Protein elution in Size Exclusion

From: Allan Pang <a.pang@qmul.ac.uk>
Date: 28 August 2011 10:25
To: CCP4BB@jiscmail.ac.uk
Hi there everyone,

What does it mean when you have proteins eluting in almost the whole column volume of S200?

I ran a gel with fractions from 8ml to 20ml and saw band for my protein all throughout.

Judging peaks on chromatogram is not useful as it doesn't have any aromatic rings.

Cheers,

Allan

--
Allan Pang

PhD Student

G35 Joseph Priestley Building
Queen Mary University of London
London
E1 4NS

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From: Roger Rowlett
It possibly means:
1. Your sample application volume was too large
2. Total protein quantity was too large
3. Fraction size was too large
4. Sample was too crude (GEC is a finishing step, not a starting step for protein purification.
5. Sample was too concentrated/viscous
6. Flow rate was too fast
7. Ionic strength of buffer too low to suppress ion exchange effects.
Roger Rowlett

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From: David Briggs
Following on from Roger's fine suggestions:

8. Your column is knackered. Can you see fine lines or cracks in the
column? Good packing is v.important for SEC columns.

HTH,

Dave


============================
David C. Briggs PhD
Father, Structural Biologist and Sceptic
============================
http://manchester.academia.edu/DavidBriggs (v.sensible)
http://xtaldave.wordpress.com/ (sensible)
http://xtaldave.posterous.com/ (less sensible)
Twitter: @xtaldave
Skype: DocDCB
============================

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From: Nian Huang
Hi, David,
What is the common cause of knackered SEC column? Will equilibrizing a buffer containing 150 mM NaCl directly into a 20% EtOH or vise versa cause the problem. There was no problem just after packing the column.

Nian

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From: David Briggs
Hi Nian,

It can be a number of things - but typically, air getting into the
column, or a leaky seal somewhere letting it dry out.

Switching from 150mM NaCl to 20% EtOH directly shouldn't cause a problem.

The reason I suggest it is that I have had the same situation as
described in the original post (protein comes off across entire volume
of SEC column elution) and it turned out the column had a leak and had
dried out in places.

Repacking the column solved the problem.

Cheers,

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From: Filip Van Petegem

worst-case scenario for crystallization purposes:

9. Your protein runs as a mixture of monomers, dimers, trimers, whatever-mers...

Filip

Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

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From: Mario Sanches
Testing for cracks in the column is quite easy. Take whichever standard you happen to have (lisozyme, GST, etc) and do a run. If it comes out in a single peak the problem is with your prep. If it comes out throughout the run the problem is with your column.

Mario Sanches

--
Mario Sanches
Postdoctoral Fellow
Samuel Lunenfeld Research Institute
Mount Sinai Hospital
600 University Ave
Toronto - Ontario
Canada
M5G 1X5
http://ca.linkedin.com/in/mariosanches

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From: R. M. Garavito

Nian,

Before you dump the column, clean it and run some protein standards on it.  If everything looks OK, run a small sample of your protein again.  If it behaves the same way, then you may have a protein with hydrophobic patches.  Anomalous binding to and elution from polymeric SEC media (sepharose, superdex, etc.) occurs with hydrophobic proteins, including some membrane proteins.  This is how they discovered hydrophobic chromatography. If you equilibrate your column in low salt, but your protein is in high salt (as might happen right after a Ni-chelation column), some of it will bind to the column.  As the salt concentration decreases, it will release off the column, spreading the protein peak over a wider volume.  However, it may still weakly bind to the column matrix and spread out even more.

If this is the case, some remedies would be to vary the salt concentration (lower the better), add in detergents, or use a chaotropic salt like LiCl instead of NaCl.

Good luck,

Michael

****************************************************************

R. Michael Garavito, Ph.D.

Professor of Biochemistry & Molecular Biology

513 Biochemistry Bldg.   

Michigan State University      

East Lansing, MI 48824-1319

****************************************************************

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From: Allan Pang

Thanks everyone for your response.

The most likely answer to my problem is protein overloaded onto the column. I pushed my protein concentration further down to 0.5ml instead of the usual method, which to run multiple times on SEC.

Adding NaCl in the buffer may also help, as it seems that the broadness of the elution was affected by addition of NaCl.

I don't think the column is knackered because the previous (and succeeding) runs of other protein samples were okay.

Different -mer state is a possibility, but something that I am not really leaning towards to, since, I managed to purify the protein before in a good elution size.

Science mystery solved.

Thanks again!

Allan

--

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From: Roger Rowlett

I'm glad this worked out well for you. For conducting molecular sizing determinations with a calibrated GEC column, I typically use 100-500 uL injections of approximately 1 mg/mL or so of purified protein on a 1.6 x 60 cm column. The limited protein quantity will prevent saturation of the internal space of the GEC media, and the limited concentration minimizes vicosity effects which hinder mass transfer between the stationary and mobile phases. At least 100 mM NaCl is important to prevent residual ion exchange interactions with the gel medium. I once worked with a protein that completely "disappeared" on a Sephadex column--lots of protein in, no protein out--at low ionic strength. When the column was washed with 500 mM NaCl, the protein magically re-appeared. Usually 100 mM NaCl is sufficient to prevent this occurrence.

Interestingly, I had a very similar conversation about GEC with a colleague and research collaborator just a couple of weeks ago. The apparent MW of our protein sample was all over the map until the sample size, concentration, vicosity, and eluant ionic strength were properly controlled, then the apparent measured MW was consistently within 10% of the calculated mass of the holoenzyme suggested by crystallography. This is typical. Keep in mind GEC measure molecular VOLUME and not MW, but most of the time we make certain assumptions that make apparent MW measurements "close enough for government work." Pay attention to the exclusion limits of the medium as well. Proteins that elute close to the void and total volumes will have poor precision in either the calibration curve or unknown determination.

Cheers,

_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 1334

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From: Nian Huang
Thanks, David. I believe the leaked column was probably my case,
although I didn't see any visible leakage I re-tightened connections
anyway. But the weird thing was that after I equilibrized the column
for a while, the dry patches and cracks disappeared. Everything
returned to normal even a standard ran fine. That's why I suspected
the mixing of reagents in the column has something to do with it in
the beginning.

Nian Huang, Ph.D.
UT Southwestern Medical Center