Large scale protein purification - Yellow and cyan fluorescent protein (Sep/16/2005 )
Hi,
Has anyone purified fluorescent proteins before? I have cells that are overexpressing CFP (cyan fp) and cells overexpressing YFP (yellow fp). I have been purifying them on a small scale using the hydrophobicity column from Bio-Rad. However, the purification will only give me 750 ul and I'm looking to get lots more than that - somewhere between 100-500 ml. It's just absurd to repeat the purification with multiple columns..I would have to do hundreds of them!! Anyone has any experience with fluorescent protein purification? I would be delighted to get some suggestions.
The following link might help.
For volume <70ml
http://www.millipore.com/markets/bioscienc...proteinresearch
For volume <500ml
http://www.millipore.com/catalogue.nsf/docs/C1975
Thanks a whole bunch! But we're looking on something that does not require anymore new equipment...something that costs less. But the site definitely gave me some idea. Thanks!
In one of my grad school rotations I purified both CFP and YFP with his-tags using a denatured protocol - lyse the cells, add GuHCl up to ~ 6M, run it through a Ni affinity column, elute and dialyse. These particular proteins are stable enough to fold on their own without chaperones.
To convert to a system like this, you'd need to reclone them into vectors that express his-tags (minimal cost, assuming you have the enzymes - you can request the vector from a published source), some Ni-NTA agarose resin (fairly cheap as reagents go, but not a minor cost), and a whole bunch of Gu-HCl (something like 30$/kg, less if you order more of course).
Out of curiosity, why are you looking for 100-500 ml of protein? Is that a typo? Usually you look for protein in the "mg/ml" or "micromolar" range, then dilute to however much you need. Are you just making a huge stock, or is it a new application?
To convert to a system like this, you'd need to reclone them into vectors that express his-tags (minimal cost, assuming you have the enzymes - you can request the vector from a published source), some Ni-NTA agarose resin (fairly cheap as reagents go, but not a minor cost), and a whole bunch of Gu-HCl (something like 30$/kg, less if you order more of course).
Out of curiosity, why are you looking for 100-500 ml of protein? Is that a typo? Usually you look for protein in the "mg/ml" or "micromolar" range, then dilute to however much you need. Are you just making a huge stock, or is it a new application?
Thanks! But it would be a lot better if I don't have to go through the cloning steps again since protein purification is not my main study. Furthermore, we don't have any vectors with his-tag and we don't do any protein purification stuff in our lab either.
I am trying to measure these two proteins in different bacterial species as a measure of ribosomal translation efficiency. However, I need to have a standard for these two proteins to be able to get an approximation of the concentration. Unfortunately, I've tried looking around for companies selling purified CFP and YFP but I couldn't find any. Clontech sells GFP but that would have a different excitation and emission wavelength, which will just complicate the calculations later. So I have to purify it myself and get a large amount of it so that I don't have to do it everytime I need a standard.