Hi there,
The purification scheme always uses the most selective chromatography in the first move in order to get rid of most of the contaminants. If this step is efficient then you get pure tagged protein. To avoid any interference of the tag with the biological activity of the purified protein, its removal is necessary most of the time by protease digest. Finally gel filtration has a triple interest: get rid of the tag and protease, check protein homogeneity (separation of oligomers and aggregates) and equilibrates the protein in a defined composition buffer.
Hi again,
There is no actual bibliography about this, it is just based on the properties of each chromatography technique and their application to protein purification.
Dear Ligar
Do you know how to concentrate sample after affinity chromotography.
e.g. I use Ni-NTA with imidazole resin column to bind His-lined fusion protein, then wash twice and elute once with DDM, PBS and imidiazole. At this moment, I obtain my eluted sample, but how to concentrate them? Or what does "concentrate sample" mean?
"concentrate sample means that to run affnity purification again?
Hi again,
there are filtration devices (Vivaspin for instance) allowing to concentrate samples using centrifugation. The cut off of the membrane has to be lower than the apparent MW of your protein of interest. In case of membrane protein, the size of the micelle has to be taken into account.
They are not the same: Vivaspin separates only two categories of proteins (ie. the ones able to go through the membrane from the ones unable to do this) whereas separation is effective for any protein within the resolution range of the gel filtration matrix. Furthemore Vivaspin allows to concentrate the retentate which is most of time the fraction of interest whereas gel filtration is always diluting due to diffusion of molecules during the chromatography process.
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