SEC for basic protein - column, buffer (Jul/18/2006 )
Hi all,
I am trying to separate high molecular weight impurities (aggregates) of a basic protein (pI around 9.4) using size exclusion chromatrography. can someone suggest to me what matrix / column or mobile phase i can use for this.. it wud be a great help!
plz tell me quick!
Thanks!
You need to tell us the approximate molecular weight of the interested protein and contaminant proteins. If you have proteins with MW close to your target protein, then SEC may not have the resolution you want to do the job. Sephacryl 300 or 500 with 0.5 M NaCl as elution phase maybe something to think about. Detailed information can be obtained from Pharmacia, now AP Bioscience.
Have you tried/considered ion exchange column, CM-Sepharose 4B, for example, for your protein?
Have you tried/considered ion exchange column, CM-Sepharose 4B, for example, for your protein?
Thanks for the reply.
My protein is ~17 kDa, and the impurities varying from 19, 20, 25 to 30 kDa. I hope to separate atleast the higher ones if not the near ones. I have tried a Silica TSK column and a superdex column, both show a high protein-matrix interaction and hence a minimal recovery of the protein (i wud say hardly anything eluted). Mobile phase used had a pH of 7, with some 0.1 M NaCl. Since the protein has a pI of 9.4 any pH above it wud make it positive and in turn make it interact with the negative matrix. My system (LC system) does not allow me to go for a higher than 8 pH, so i can work in the later range.
I shal try looking up pharmacia too.
Do tell me if u have any further suggestions.
No, i havent tried any ion exchange chr. on my protein. U have any suggestions on that?
Thanks a ton!
Increase ionic strength to 0.5 M can reduce nonspecific interaction, but it is resin dependent. From what you have said SEC probably is not the best choice for you, as your interested protein will come out at last and gets very diluted. Since your protein has small MW, (NH4)2SO4 precipitation would also be a good initial step to get rid of a lot of junks. Afterwards you dialyse it and you may go with ion exchange column. There is no universal purification stratigy for a particular protein. It takes trial and error. I suggest that get a book and start to read about it more before waste your time.
Oops, sorry but my purpose is not purification but is to resolve and quantitate these impurities, analytical. i may in future want to remove them. but i need to do an SEC for all that first.
Ya, plz tell me how ion exchange will help.. these high mol wieght impurities are very related.. i mean mostly same but a few amino acids synthesized over the stop codon. So most properties except for size r similar to a very high extent. so i think i need sec for this reason too.
Plz do suggest.. thanks a ton!
PS: increased ionic strength would increase my recovery.. can u plz explain how?