I am new to handle intricate protein purification, now going through the problem of concentrating my protein. I did His-trap -> dialysis -> DEAE-trap -> dialysis at the end I had something like 10 ml of dialyzed protein at a conc. of 0.17mg/ml. For the enzymatic assays I planned to do it would be much better if the conc. is at least 2mg/ml. With PEG 35000 I already reduced the volume to 3 ml. I am too scared to leave it in dialysis bag just because one can't see the volume reduction that clear in them. I transferred the contents from the dialysis bag to 3 eppis and tied their mouth with dialysis membrane, leaving the tubes upside down on a bed of dry PEG. I see the volume continue to reduce but at very slower rate, mean while the membrane is also getting dried.
now my worries are:
1. Will the concentrating process be still effective like this.
2. Is there any other better ways (except amicon, acetone precipitation- I just don't want to loose protein in those, I am reading a lot of issues around them here in bioforum, and lyophilizer- as we don't have a refrigerated one) to concentrate protein without loosing its native activity.
3. All the processes I mentioned above to extract and purify my protein took 1 week, since the day 1 they are in 4 degrees, and I don't know when this concentrating process will end. How much likely that I loose my protein for degradation, and to what extent.
condition I think I can't change: is the initial volume of cell lysate. I already start with a viscous extract even after DNase treatment.
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