I am purifying a 20 kDa recombinant protein. Protein concentration is greatly reduced after dialysis. My dialysis buffer is 20 mM Tris buffer pH 8.0 that contains 150 mM NaCl, 10% glycerol. How can I prevent the recombinant protein from aggregation during dialysis?
Dear Hossein
which was the original buffer (before dialysis)? Why did you add 10% of glycerol?
Generally i prefer perform rapid buffer exchange by desalting instead dialysis. In this way you can and add glycerol (which has high density and can slow down the buffer exchange protocol) at the end of rhe buffer exchange.
IF you are interested to know more detail about desalting you can give a look to the following videos:
https://www.blogger.com/video.g?token=AD6v5dzeX21U_Er6jBChQIhE5zWtQhNjRjNXRPR7qk7KfWWSQxhf8PbUn4R6KLL0pn_7tU92M0-B3kEUHO9IkTpelmdO79QR53uSg29ZFV3j5D-siHAamTu1y2wOEQ8S4As_wpxzLvA
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
avaialble on my blog: ProteoCool
ciao
Manuele
Hi Hossein,
Why did you use the dialysis method to purify the target protein? In my previous experience, the dialysis process tends to cause protein aggregation. I usually use gel filtration to purify the protein and for rapid buffer exchange.
Dear Roger Davis
I do not have gel filtration facilities and equipments.
Hossein Ansariniya Cellulose (C) membranes are recommended for classic uses for proteins below 20KDa or for quicker dialysis kinetics.
Perhaps pH of buffer solution is high.l hope following study will help you
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