During protein concentration step by using Amicon tubes with 10KDa cutoff (from Merck company), I could see some cloudy suspension which indicate for protein aggregates formation. Afterwards in SEC (GF) step I had an elution profile with low resolution due to these aggregates which came in the void peak and start smearing as a shoulder for the void peak in the chromatogram.
I use DTT (1mM), EGTA (1mM), NaCl (50mM), and MgCl2 (1mM) in Lysis, wash , and gel filtration buffers
Although very common due to its simplicity, concentration with centrifuge devices is possible the worst method available due to the extremely high local concentration you obtain at the membrane: many proteins aggregate or denature at such high concentrations. More delicate concentration methods are stirred devices (e.g. Amicon) or Tangential Flow Filtration or TFF (e.g. Sartorius).
Additionally, never inject dirty material on a chromatographic column, you should filter before the actual purification step, with 2 advantages: 1. remove the aggregated protein, and at the end of SEC obtain cleaner material; 2. prevent damage to your SEC column.
See to what extent you can concentrate without losing too much protein. After concentrating, collect the protein in a centrifuge tube and centrifuge. Load the supernatant. This way you minimize loading visible aggregates to some extent thus helping your column at the very least. Alternatively, you could go for syringe filtering with a 0.22 micron filter. Your chromatographic column is precious, so always try to load as clean a sample as possible. Of course some amount of soluble aggregates will also be still there most likely. So I believe you will get a peak in the void volume still. But your resolution for expected/ desired -meric state of your protein might improve. This is just a suggestion. See if it works.
As others have pointed out, the long run approach will be for you to work out the buffer conditions where your protein is happiest. For that you need to play around with your salt strength, buffer pH and so on so forth.
Although very common due to its simplicity, concentration with centrifuge devices is possible the worst method available due to the extremely high local concentration you obtain at the membrane: many proteins aggregate or denature at such high concentrations. More delicate concentration methods are stirred devices (e.g. Amicon) or Tangential Flow Filtration or TFF (e.g. Sartorius).
Additionally, never inject dirty material on a chromatographic column, you should filter before the actual purification step, with 2 advantages: 1. remove the aggregated protein, and at the end of SEC obtain cleaner material; 2. prevent damage to your SEC column.
Hi there,
So you have to adjust the buffer condition so that your protein is more stable in it when concentrated. The parameters to optimize are : nature of the buffer, pH (what is the pI of your protein? pH has to be at least one unit away from pI) and ionic strength (50mM being rather low). You may also consider additives like glycerol for instance.
Did I understand correctly that you are using gel filtration as a first purification step after lysis? I agree with Carlo that spin filter concentration is a harsh treatment of the proteins. I would try a concentrating technique such as ion exchange as the first purification step. Never inject cloudy material on the column - especially a gel filtration column.
I agree with Dominique. The pH of your buffer may be to close to the protein pI.
Also, why does your buffer have EGTA AND Mg++?? Plus no detergents like octylglucoside.
Since you used 1 mM DTT in all your buffers, I assume that your protein has some disulphide bonds. DTT is not a stable compound and easily inactivated over the time at the pH 7.5 and above. If at any step you used not freshly-prepared DTT (including GF buffer) your protein might form some insoluble disulphide oligomers or some disulphide complexes with other proteins during gel-filtration. That might explain the observation you described.
Re: Joey Lee - Tm and aggregation during concentration are not necessarily related, furthermore in my times all purifications where carried out in the cold room unless there was a good reason not to. Glycerol may or may not help preventing aggregation, and you have so many other options that could be as useful on an individual protein basis. But why bothering screening all sorts of additives when you have better concentration methods available?!
1>At a pH of 7.5 MgCl2 hydrolyses to insoluble floculent hydrated magnesium hydroxide and oxide.EDTA doesn't complex Mg2+ below pH 11 so its not protecting.
2>As everyone has said be careful of your centrifuge speeds so as not to centrifuge out the protein
3>If u got a cloudy solution leave it to filter overnight and leave the lab, next day when u get the residue u can see if its acid soluble Mg(II) or do a Millon's/N inhydrin test.Ninhydrin test of course responds if some aa are present.
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