2 replies to this topic

[Candice]

I have used thrombin to cleave my GST-tag fused protein in the buffer 500mM NaCl, 500mM urea, 20mM HEPES, 1mM DTT, pH 7.4 after elution from GST column. It is discovered that the thrombin activity is very low. Does reduced gluathione affect the activity of thrombin? Thanks a lot!

[Kaioshin]

Candice, on 03 May 2011 - 07:54 PM, said:

I have used thrombin to cleave my GST-tag fused protein in the buffer 500mM NaCl, 500mM urea, 20mM HEPES, 1mM DTT, pH 7.4 after elution from GST column. It is discovered that the thrombin activity is very low. Does reduced gluathione affect the activity of thrombin? Thanks a lot!

If your thrombin activity is low, I would guess it is the UREA causing problems, though that's just a guess

[xabiarias]

I used to succesfully cut with Thrombin my fusion protein in Tris Buffer with 10 mM reduced gluthation !