HPLC Anion Exchange Protein Standard
Posted: Mon Oct 24, 2011 12:30 am
Hi all,
I’m new to the board and new to liquid chromatography as well. After almost a year, my project is reaching its protein purification stage, so over the next year I’ll be doing a lot of HPLC purification. I was put in charge of getting our HPLC running which is kind of overwhelming!
I have a small Bio-Rad DEAE anion exchange column. The first thing I was told to do was find the protein standard and the chromatogram for the standard and to reproduce the chromatogram. Turns out we don’t have either, and it costs $200 to buy! Is there any way I can make up my own “standard” mixture of proteins to test? Could I just make a mixture of maybe 4 proteins with different isoelectric points, do HPLC, and see if the peaks elute in the order I would expect? We have a bunch of common proteins sitting around (BSA, IgG, cytochrome C, etc.)
Or, could I run a mixture through an HPLC that I know is working properly (hooked up to our DEAE column), and then run an identical mixture through our HPLC with the DEAE, and see if the chromatograms are the same to verify that our HPLC is working?
Is there a better way to go about this, or could my ideas work? Right now, I don’t care about anything quantitative- I just want to be able to get a nice separation, and to be able to reliably reproduce that separation.
Thanks for your help,
A confused biologist
I’m new to the board and new to liquid chromatography as well. After almost a year, my project is reaching its protein purification stage, so over the next year I’ll be doing a lot of HPLC purification. I was put in charge of getting our HPLC running which is kind of overwhelming!
I have a small Bio-Rad DEAE anion exchange column. The first thing I was told to do was find the protein standard and the chromatogram for the standard and to reproduce the chromatogram. Turns out we don’t have either, and it costs $200 to buy! Is there any way I can make up my own “standard” mixture of proteins to test? Could I just make a mixture of maybe 4 proteins with different isoelectric points, do HPLC, and see if the peaks elute in the order I would expect? We have a bunch of common proteins sitting around (BSA, IgG, cytochrome C, etc.)
Or, could I run a mixture through an HPLC that I know is working properly (hooked up to our DEAE column), and then run an identical mixture through our HPLC with the DEAE, and see if the chromatograms are the same to verify that our HPLC is working?
Is there a better way to go about this, or could my ideas work? Right now, I don’t care about anything quantitative- I just want to be able to get a nice separation, and to be able to reliably reproduce that separation.
Thanks for your help,
A confused biologist