how should I concentrate my protein?? - (Apr/18/2005 )
i have a very dilute solution of my protein(~ 14kDa) and i tried concentrating it with centricon filters(MWCO-3kDa). but i do not recover my protein. i dont know what the problem can be??does the protein precipitate??
i want to concentrate the protein to do an in-gel digestion followed by MALDI.
would appreciate any suggestion.
If your protein is hydrophilic, I think your protein is bound to the dialysis memabne and you need to wash it off vigrously. However, to resolve whether your protein is being flowing through the membrane you can use the flow through and comapre it against the solution remaining in the centricon cup in western blotting experiment (that is if you have an antibody against it).
thanks for ur suggestions. my protein is quite hydrophobic. any comments on this aspect?? is TCA precipitation suitable if i dont want to use the filters??
Yes TCA would be a good way to concentrate it, good Luck!
can i concentrate using chloroform/methanol precipitation??
I am not sure about, Chlorform/Methanol, but I have used in the past ice cold acetone and ice cold ethanol as a method of protein concentration and both worked for my application. I will ask my colleagues around here if they have used Chloroform/Methanol, meanwhile Good Luck!
This link has a very good article in this regard, Enjoy!
Common methods of protein concentration pursued in our lab are:
Do not use TCA, acetone, or methanol concentration. these methods will denature your protein, so not very suitable for structural analysis.
then which method would u recommend?? i can´t use the cetricon filters.