His tag protein purification - (Mar/23/2011 )

Hi all,
I am a newbie to protein purification. I am trying to purify a phospholipase from S2 cells. Initially I did the purification manually by making my own columns with Ni-NTA resin. I carried out the purification at 4 degrees. Although it did not turn out to be quite pure but it showed good activity and from the native cells I found out that there was no background activity. Recently our lab bought an Akta which has been placed in room temperature. I carried out the purification first with Ni and then with an anion exchanger. The purity was very good. But finally when i checked for the activity it was relatively very very low from the Ni-NTA fractions as well as the ion exchanger fractions. I carried out the purification a few times but the result is the same. I have 20% glycerol in all the buffers. I cant figure out what the reason might be. The only thing i could think of is the temperature. This week I am moving the Akta into the cold room. Can anyone tell me if temperature could be the main cause or anything else other than that.

Please can anyone give me some suggestion.

Regards.

temperature may be your problem.

i wouldn't put the akta into the cold room. instead you can use your buffers cold (keep them in an ice bath) and ice the collector tray so the fractions stay cold

I did try that but not much change in the activity....just a slight bit of increase....Do u think placing the akta in the cold room would harm it. If I place the Akta in the cold room and carry out the purification would there be any difference in the end result.

I did try that but not much change in the activity....just a slight bit of increase....Do u think placing the akta in the cold room would harm it. If I place the Akta in the cold room and carry out the purification would there be any difference in the end result.

if you place the akta in the cold room then you can never take it out (you can but it will require a lot of maintenance) or you will void any warranty. you also have to hope that the cold room never breaks down.

you can also jacket your column and run cold water through it if chilling the buffer and fractions is not enough (it should be, if the protein is still losing activity then something else may be happening to it, not temperature related).

Hi I tried all that you have mentioned but everything was in vain. Now I am really tensed...I have been working for ages on this and have repeated many many times and now I am in a state where I have lost interest...I really dont know what to do...!!! I have also moved the Akta into the cold room and tested with it but to no avail. The result is the same...!!!

Please some one help me.

kcchalmers on Wed Mar 23 16:09:25 2011 said:

your problem may be concentration dependent. some enzymes must be at or above some critical concentration to be active.

another possibility is that your protein may not like being pressurized. the drop in pressure when it exits the column (or exits the tubing if you have a back pressure regulator) may be denaturing your protein.