Hi fellow researchers, am an newbie in protein purification and hence this question. I am working on a novel protein whose size is 17kda.it does not have cysteine. It's PI is around 7.83. I did a his tag to purify it. I did able to purify.. But when I want to remove imidazole it aggregates I guess. Last time I could able to recover 2mg of my protein for a litre of culture. This time nothing I got.. When I diid sec using s200 column, everything gets eluted close to void volume (2ml away from void). But its single mono dispersed peak. I use ph 8.5 tris buffer . could anyone tell me what could be the possible problem?
Your method sounds reasonable. It's not necessarily your fault. Some proteins do that.
You should try a bunch of different conditions, to see if you can find a SEC buffer that prevents the aggregation. There is no good way to predict what might work. The next few obvious things to try would be:
- Use an acidic buffer, for example ammonium acetate, or pipes, pH ~6.5.
- Add salt to 500 mM.
- Add detergent, like 0.1% triton-x-100 or igepal-ca-630.
See what that does....
Is it important that you remove imidazole from your buffer? So,etimes keeping a small amount 5-20 mM imidazole can help stabilize some proteins.
Another trick is to slowly dialysis out imidazole by using a dialysis membrane. Also, try changing your salt concentration.
When you say aggregates, do you see visible precipitation or is this an assumption? Maybe your protein forms a Multimer?
His tag purifications are best done in phosphate buffer around pH ~7.5 so the His tag is negatively charged, but you might have problems with this because pH 7.5 is so close to your protein's pI. What pH did you use? The imidazole is best made up fresh and its quite basic and needs HCl addition to reach a pH ~8.0 before use. Is it possible the pH of your purification/elution was different? If your pH was further away from your proteins pI on the first (successful) purification but the pH was too close on the later purification this might explain the precipitation. I'd double check the pHs!
John Schloendorn , Thanks a ton for your input. I had plans to increase my salt in the SEC buffer. But I dont want detergent, as I have to do some invitro assays downstream.
Again Swapna Aravind , Thank you for your time. Am not sure if imidazole is good for the assays I have planned further. Swapna, I dont see precipitation as such when I spin too.. So it could be multimer.. How can I know this? If its multimer will it dissociate at a suitable ph?
Edward Michelini: Thank you . Tris is capable of changing temperature and I did my purification at room temperature as I know my protein is stable. at 4 degrees the buffer which I made would not reach close to my proteins PI. anyways I will check this again. Thanks a ton fellows.
Dear all.,
Thanks for your input. I have increased sodium chloride concentration in my buffer and it solved my problem.
Thanks.
Revathy.K
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