05Feb-09Feb18

06FEB18

IRM13

I sonicated some cells (a small amount of cells) so I can finish off the IRM13 experiments (the ions activity enhancement). I dialysed some of the CFE and kept some non dialysed.

I repeated the experiment on page 56 at DR1117-073 doing exactly the same thing for t=30min only but this time I didnt add any substrate in the buffers. There is a suspect that the ions (and especially iron) acquire a color by themselves and the absorbance recorded is due to the ions in the buffers and not due to the PNPG hydrolysis (and nitro-phenyl production).

I repeated the experiment then with no substrate in. Results in page

I also repeated exactly the same experiment with ions and IRM13 CFE dialysed and non dilysed but this time I used 5mM of ions (in case that the concentration of the ions was high. I did this experiment with 5mM ions with substrate and without substrate as a control. Results in page


IRM12

I also sonicated some cells and dialysed some of the CFE while kept some CFE non-dialysed.
I did the experiment on page 55 in DR 1117-073 for checking about activity between the non dialysed and dialysed CFE. The dialysed CFE is equally active to the non-dialysed. This means that I will probably be able to continue with the characterising of the IRM12 (possibly after size exclusion separation first).

07FEB18

I sonicated a lot of IRM12 cells, produced the CFE, used AKTA. The column got air in and I could continue the protein production. I left the column overnight for getting moist again drop by drop.

08FEB
In the morning the column was fixed and no air was in. However, my protein was left in room temperature overnight and I dont know whether is still active. I did AKTA elution (using the method Elution_08FEB18) and saw a pick at F6, F7 and F8 where the buffer B was at 20% (which means 100mM Imidazole). I put these 3 fraction for dialysis with 2lt of buffer A for 3 hours. I check all of the fractions on an SDS gel for detecting my protein. I should be aware that in size exclusion gel on the 5JAN18 the protein was found to be 120kDa (see gel and western on pages 52, 53 in DR 1117-073). So I might see my protein higher this time as well.

I pulled the fractions so that I check 1-3, 4-5, 6, 7, 8, 9-11, 12-15, 16-18, 19-21, 22-24, 24-26.

The gel detected the protein in the fractions dialysed (see the gel on page      )
I will be needing to do size exclusion as the fractions are not clean. First I need to check for activity because I might no having activity to my protein and there is no point of getting into the size exclusion. I check the activity by putting 100μl of buffer+PNPX and either 1uM or 10uM ( I tested both). I left the plate at 37oC for 5 minutes but I had negative results. The protein might be destroyed because of remaining in room temperature for so many hours, might be the PNPG which was kept in the freezer, of my have lost my protein during dialysis.

Tomorrow I want to test whether my protein is in the dialysed fractions and I will also make new fresh PNPX buffer and repeate the assay. It would be good if I could also measure the level of Imidazole in the dialysed fractions




09FEB

I repeated the yesterday experiment about activity using freshly made PNPX this time but it didnt work.

I measured the pre-dialysis  and after dialysis absorbance at 280nm because Imidazole absorbs at 280nm. Pre= 0.28 after=0.5689

I also run a western with the dialysed and non-dialysed samples to detect the protein. The western didnt work is totally empty for all of the samples