After successful purification of my protein of interest by affinity chromatography (first picture, right side), yielding 50ml of 0.4 mg/ml (estimated by nanodrop), I went on to perform concentration on an Amicon Ultra 15 device. The MWCO is 3kDa, while my protein is 13KDa. We take extra care to centrifuge at low speed (2500 rpm instead of the 4000g that the supplier recommands), and to "wash" the filter every 10 minutes by up-and-downs to reduce adsorption and protein loss in the filter. Every hour, the concentration in the retentate is measured against the filtrate as a blank, by Nanodrop.
The problem is, it looks like our protein concentration is not increasing at all, even though nothing is found in the filtrate as well. An SDS-PAGE was performed to confirm that the protein is not lost in the filtrate. However, in the retentate (after a 30x concentration factor), the protein band's intensity is almost the same as before the concentration (2nd picture).
Is anyone familiar with this problem ? How can we prevent it ?
I used to find that swelling dry sephadex of a grade that completely excludes the protein size is a quick and easy method for concentration but you have to be careful not to use too much sephadex. You lose some but it may be less than you are currently losing on your membrane
Did you consider unspecific adsorption of your protein to the filter material? In some cases it helps to saturate the filter material with a protein (serum albumin for example) and subsequent washing prior to concentration of your protein.
Peter
In addition to the two previous answers, you may also try to precipitate the protein with ammonium sulfate, followed by centrifugation, disssolution of the pellet in a suitable buffer and dialysis to get rid of residual ammonium sulfate.However, it may be that your protein precipitates at high concentration. This usually leads to clogging of the filter and very slow filtration. Thus your best bet may be to try to increase the solubility of your protein, e.g. by increasing the ionic strength, adjusting the pH, etc. Detergents are not recommended, as they will form micelles that will accumulate.
Hi Martin,
I have encountered a similar situation and finally found out that I did not mix the concentrated protein solution evenly before measuring the concentration. As we all know, during the process of concentration, the macromolecules are trapped and accumulated on the membrane surface. The concentration on the surface of the membrane is much higher than that in the solution, so it is necessary to gently blow the membrane continuously after the concentration, this process may even take more than one minute.
Hope it helps!
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