After the first experiment(s) I had to leave for my winter break, so stored the enzymes in 4 degs (all in centigrade/ deg C) (As mentioned in the bottle). But my project guide told me to put it in -20 degrees as I had suspended the entire quantity of the enzyme in 0.1 Molar Phosphate buffe (K2HPO4 and KH2PO4).
It's been there all the time(around 3.5 months now)
When I use the enzyme, I have a working solution out of it by taking a small volume of it and storing at 4 degrees to use for the next week,etc.
But after the first attempt, the reaction has not worked. Except when I used around 10 KU by mistake with the substrate - This sample was stored at 4 degrees, but when I tried to make the required quantity sample (0.04 units ml-1), it again did not work.
Only once did I test with my sample, after that I have only been trying the 100% reaction( buffer + enzyme + substrate) of the enzyme Substrate reaction and it is not been working. Working = Buffer + Ez+ Sub @ 37 deg C for 30 mins = Yellow color formation from colorless solution.
The Beta glucosidase, I got in Feb this year and from the first attempt itself its not been working. It's been stored at -20 as well, like the previous enzyme.
Substrate: Beta - D - Gluco/galactopyranoside. (stored at 4 degree Centigrate)
Buffer: Phosphate Buffer 0.1 M (as mentioned above). Fresh batches prepared regularly.
Please help, my project has been stuck for almost 3 months because of this and I am not able to troubleshoot where the assay is going wrong.
Edited by metaltemujin, 15 April 2011 - 05:34 AM.