The effect of the size of a protein on choosing a purification method
Hi there,
You have to tell us more about your favorite protein:
Is it soluble/non soluble (membrane protein)?
Is it secreted?
Is the protein tagged or do you have in hands a specific antibody raised against it?
If soluble in a first move you could pass your cell lysate onto a 50kDa cut off membrane (or even 100kDa cut off if it actually retains the protein) in order to get rid of "small" proteins which are quite abundant.
Hi Dominique, It is soluble, not secreted, not-tagged, and raising Ab is not feasible in our college right now so I do not think about rasing Ab yet, though I was thinking a kind of tag purification like His-tag (using plasmids like pYES2 NT series from invitrogen) but not sure because the protein is rather large and might be difficult to be trapped on the column.
Hi again, Histag is definitely an option 100kDa being not that big considering protein purification...
Hi J!
I wonder if you have any experience with what you suggested? And what happens if gel filteration not included? is it just for having cleaner result? Please elaborate a little bit on this, if you could. Thank you very much.
gel filtration is a chromatographic technique that separates proteins in their native form according to the moelcular weight. you can read about it in books on analytical biochemistry.
unless you have a tag, protein purification always is a multi-step procedure.
because your protein is bigger than many other proteins in a cell, gel filtration is an especially useful step in your case.
Best wishes
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