inactive enzyme from purification - (Jan/09/2011 )

Hi

I am purifying an enzyme with GST-tag; however, my enzyme is not active. I have a few questions about this.
1. I wondered how long I can store the pellet (after I harvest the cell)
in -80C or -20C freezer before doing the purification. Is this affect enzyme activity?
2. My enzyme requires Mg2+ for functioning. Do I need to add Mg2+ during expression, induction or purification?
I cannot find this detail in the papers.
3. I also added 0.1% triton X100 in lysis buffer to help my enzyme soluble. Is triton X-100 able to inactivate
an enzyme?

Waiting for suggestions and thank you in advance

winter11 on Mon Jan 10 05:33:46 2011 said:

some enzymes can handle freezing, some can't. some will survive if not actually frozen (eg-in solution with up to 50% glycerol). you have to determine if yours can.

usually, the mg is required when the enzyme must be active (eg- in an assay). sometimes a cofactor is required for stability. you can find out by looking at literature about the isolation of the enzyme.

some enzymes can and some can't handle the presence of some additives. again, something you need to determine for your enzyme.