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inactive enzyme from purification


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#1 winter11

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Posted 09 January 2011 - 09:33 PM

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 :)

#2 mdfenko

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Posted 10 January 2011 - 09:09 AM

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?

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.

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.

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.

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?

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