suggest protein purification methods? - (Mar/20/2006 )
Hi,
My PI has recently asked me to come up with a plan for starting a protein purification core. I have never done Protein purification before so I am in the reseach phase now but I am planning on using NiNTA resin in 20ml columns and a two step column protocol. Can anyone give me any suggestions on what is the best protocol to use and where I can get reagents and materials at an economical price? I have heard that the beads are not good to use because they are very expensive and do not give a good yield. Any thought on this?
Cheers,
Jodi
My PI has recently asked me to come up with a plan for starting a protein purification core. I have never done Protein purification before so I am in the reseach phase now but I am planning on using NiNTA resin in 20ml columns and a two step column protocol. Can anyone give me any suggestions on what is the best protocol to use and where I can get reagents and materials at an economical price? I have heard that the beads are not good to use because they are very expensive and do not give a good yield. Any thought on this?
Cheers,
Jodi
HI,
Are you going to purify His tagged protein? I used TALON Metal Affinity Resin from Clontech several years ago and I had very good experience with it. Pulled most of the target protein down from E.coli overexpression.
Good luck.
I agree, the easiest purification I now works with tags- I also have a His-tag on my enzyme and get very pure results after a single run over a Ni-sepharose column from GE Healthcare (former Amersham).
If your protein is not tagged yet (and you're not planning to do so in the future), you'll most propably have to do several purification steps like ammonium sulfate precipitation, gel filtration and / or anion exchange until you get your (mostly) pure protein of interest. For that, you need to know a lot about your protein- like, size (you can determine that with protein standards), charge and stuff like that.