13NOV17 Meeting with Frank

13NOV17

I showed the PNPG and PNPX graphs to Frank (see DR1117-073 page 10). He suggested to use a negative control as well as the positive I have used (B99). I need to repeat the experiment with a negative control (water in the in vitro kit). So after this meeting I have to :

Write down the list of the assays and procedure based on the yak paper
Calculate how much protein I need for the assays
Do a purification of IRM13 small scale
Run the in vitro protein production for IRM 6-13 and also for a black and B99
Run the TapeStation for IRM6-13 and also for IRM1
Run the PNPG and PNPX activity experiment for IRM1-13 with a blank
Check where the IRM12 and 13 come from (organisms)


CHECKING IRM12 PROTEIN PURITY

I run a gel using sample of the pure IRM12 protein produced from the previous purification and it seems that my protein is not pure. There is a suggestion to dilute it in 10ml buffer standard A (see the purification procedure in DR 0817-073 page 116) and put 1ml of resin, incubate it for an hour and follow the procedure again for purifying but using Imidazole concentrations from 20 -150mM. But I forgot to remove the ethanol from the resin and equilibrate it and I put 1ml of resin _ethanol in the pure protein. Ethanol denatures proteins.

SCALING UP OF IRM12 AND IRM13

I scaled up the IRM12 and 13 cultures to 800ml at 30oC with 0.1mM IPTG