Hello everyone,

I am trying to assess the specific activity of trypsin in pancreatin, according to the INFOGEST protocol. However, I am running into several problems.

We scaled the experiment down to 300µl instead of 3ml since we want to use a UV transparent 96 well plate instead of cuvettes.

First problem: when I add the recommended amount of TAME to my blank measurement, the blank is already at 1 when measuring. According to the protocol it should be close to 0.

So we are now using only 3µl of TAME (instead of 30µl) which gives an initial blank measurement of around 0.2.

When we tried it with pancreatin, we got results of below 1 U/mg. So we tried to use trypsin with a known activity and used the recommended amount from the protocol (10µg/ml, 15µg/ml and 20µg/ml) and got barely any change. So we went up to 100µg/ml and 300µg/ml and nothing changed either. We even let the experiment run for around 2 hours to see if any change would occur.

Does anyone have any idea how to approach these problems?

Thank you so much!