enzyme assay with thioredoxin

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enzyme assay with thioredoxin

Post by piefke » Fri Jul 23, 2010 12:46 pm

I want to test the influence of thioredoxin on the activity of my heterologously expressed enzyme. I incubate the enzyme, thioredoxin (3µM) and DTT (10 mM) for 15 minutes and then start the activity measurement. one time it worked well and I could detect an increasing activity with increasing concentrations of TRX. But in the last measurments I detected the same activity in my sample with TRX and in the control sample without TRX.
Does anyone have an idea?
I solved the TRX in water and freezed aliquots at -80 °C. Perhaps it is not stable under these conditions?
I use DTT to reduce the TRX. Is something wrong with mixing TRX and DTT and incuabate then for 15 minutes?
I don´t have any idea :(

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Re: enzyme assay with thioredoxin

Post by patrikstorm » Fri Apr 27, 2012 11:31 am

What kind of activity are you studying? I have observed that possible trace contaminants in DTT can cause breakdown of hydrogen peroxide that gets more severe with increasing temperature; not always though and especially not with the lower concentration (1 mM) of DTT that I used. Incubation time should not be a problem. Be aware though that DTT has a shorter halflife in solution with increasing pH and temperature (make fresh always).

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Post by JackBean » Wed May 02, 2012 8:45 am

How do you store your DTT?

Cis or trans? That's what matters.

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