Ingenza

12FEB I degassed 2lt of Buffer A for IRM12 and 2 lt of water for washing the size exclusion column and equilibrate it over night 13FEB I sonicated IRM12, got the CFE, and run the AKTA. This time I put 20mM of Imidazole in buffer A to minimise the unspecific binding I usually have (thats why the size exclusion separation is needed). I could see any pick in the AKTA graph and I therefore run all the samples on the gel and left it for staining (see page           for the gel). ***NOTE: For making the SDS samples INSOLUBLE FRACTION: 10μl sample (as usual following the recipe) CFE: 5 μl sample + H2O AKTA FRACTIONS: 25μl sample I load 10μl of the Insoluble and CFE while I load 20μl of the AKTA FRACTIONS 14FEB Before using the size exclusion I need to be sure that my protein is the one I see on the gel (page       ) So I run a western using the samples from yesterday. The IRM12 his tagged protein was found in the last AKTA...

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 kep...

02FEB18 There is an idea about the NaCl concentration in the dialysis mixture. The NaCl concentration is 500mM which is higher than the inside the bag NaCl concentration which means that NaCl gets into the bag during dialysis. I need to check this Also I need to check whether Imidazole gets out from the bag after dialysis. To check than I need to measure the Imidazole concentration at 280nm before dialysis and after. Also measure the concentration of protein before dialysis and after. Dropping the ions concentration (might be high) Repeat the experiment with the ions and no substrate as control