I have successfully purified my 13 kDa protein of interest using an Ni-NTA chromatography (see image attached which are my elution fractions). I want to further purify and concentrate it.
It seems like Amicon columns are not compatible with high imidazole concentrations like I have in my elution fractions (200mM). What are your suggestions to remove high weight proteins, change the buffer and concentrate my sample ?
Martin Chenal
If you want to concentrate it even more, you can do a TCA protein elution.
Firstly you could do dialysis to remove the imidazole. Then to further purify the protein, I would recommend ion exchange and/or gel filtration.
For ion exchange, you can dialyse into a low salt buffer without concentrating and load that onto the column (IX is a concentrating step). For gel filtration you would have to concentrate the sample before loading it on.
I always go for GF last as it gives information about the oligomeric state/aggregation in the protein prep.
Gel filtration is also a low capacity step. Ideally, you do not apply more than 3% of the column volume onto a gel filtration column; ion exchange columns have no volume limit.
I was thinking of using Zebaspin desalt column (4ml max volume, MWCO of 7kDa) to purify and change buffer, and then maybe an Amicon column (3kDa MWCO) to concentrate my sample. Does that sound ok ?
Also, would it be possible to use an Amicon column of 30kDa MWCO and collect the flowthrough instead of the retentate, in order to remove unwanted high MW proteins ?
Hi Martin,
Depending upon your sample volume you can either dialyse your sample against a buffer containing no imidazole, or you could use a commercial desalting column (see here: https://www.gelifesciences.com/en/gb/shop/chromatography/prepacked-columns/desalting-and-buffer-exchange )
Following this step you can concentrate your sample in an Amicon concentrator, and then apply it to a SEC (although centrifuge/filter first). SEC is itself a buffer exchange step, so if you have only a few mL of sample you may be able to just apply your sample directly onto the column without bothering with dialysis/concentrating.
Remember to centrifuge your sample thoroughly prior to application to the SEC, as any aggregates will clog it up. (In my experience filtering - even the 'low-bind' kind - reduces your yield).
Hope this helps!
Best wishes,
James
As James Torpey mentioned, you will always get protein biding to a membrane. To minimize the loss, after removing the concentrate, the membrane is usually washed with a small amount of buffer. But this will naturally reduce the concentration factor.
You mention using a 30kDa filter. But your protein is only 13kDa. Your higher molecular multimers could easily fall apart during concentration and you would lose all protein. Therefore, a 10kDa filter is the largest you should use.
Looking at James' post, I would make the following change.
Concentrate your protein mix with a 10kDa concentration device. Do at least 2 washes of the membrane with running buffer - pipetting the buffer sitting on the membrane a few times into a pipette and back onto the membrane to loosen any bound protein. Best would be for the first wash to add buffer onto the membrane and wait ~10min to give the bound protein a chance to dissolve, then do the pipetting.
The concentrate centrifuge hard and then apply to a SEC column. As James mentions, the step will automatically exchange the sample buffer against the running buffer. So you do not need an additional buffer exchange step.
As a general rule, the cut off size of a membrane should be about 30% of the mass of your protein, so for a 13 kDa protein I'd use a 3 kDa membrane to ensure maximal protein yield at a filtration rate as high as possible. Once you have reduced the volume to your liking, remove the pressure and then continue to stir the cell for 20-30 min, this will get as much as possible proteins off the filtration membrane.
You can do a buffer exchange in an Amicon cell, just concentrate, add the new buffer and concentrate again. If necessary, repeat.
Engelbert Buxbaum, yes, 30% of the MW is the optimal choice to come as close as possible to 100% yield. But except for large proteins, like antibodies, I do not know anyone following this rule, as the small pore sizes can slow down the process quite significantly.
I try to stay 30% to 40% under the MW or the protein of interest and accept a certain loss.
Hi Martin,
If your sample volume is less, I recommend using an ultrafiltration membrane system with MWCO of 3kD, but the concentration process may be slow. And I recommend that you purify the target protein through further operations, such as gel filtration or ion exchange.
All the best!
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