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

22JAN18 I made the sonication of IRM13 and when produced the CFE I checked for activity. I have noticed that IRM13 elutes at about 45% buffer B (500mM Imidazole) and 65% buffer A (0mM Imidazole). This means that IRM13 elutes at 225mM approx and on (there is some unspecific binding before the 225mM and continues the elution up to 500mM Imidazole). So I made phosphate buffer pH 7 with 1.25mM PNPG and PNPX containing 225mM Imidazole and tested the activity  of the CFE. Also I used phosphate buffer pH7 + 225mM Imidazole without substrate as a control. I placed 100ul of buffer+substrate+imidazole in the wells and then 25ul of CFE with the multi-channel pipette in 1 and 2 column. I incubated for 5 min at 37oC, quenched the reaction with 50ul Na2CO3 and measured the absorbance 405nm. See the diagram of the plate on page The values are significantly increased compared to previous week that I measure the activity of the IRM14 CFE without Imidazole which means that Imidazole inhibi...

11JAN18 I started the pH assays. I made 50mM citric buffer with 1.25mM PNPG and PNPX (mixing citric acid 50mM + 1.25mM PNPG and PNPX and 50mM citrate + 1.25mM PNPG and PNPX) for pH range 3.5-5.5, phosphate buffer 50mM (mixing phospate monobasic 50mM + 1.25mM PNPG and PNPX and phosphate dibasic 50mM + 1.25mM PNPG and PNPX) for pH range 6-7.5 and Tris 50mM + 1.25mM PNPG and PNPX pH adjusted for pH 8-9. The recipes for the citrate and phosphate buffer are on page 42 at DR 0417-073. Tris was adjusted with NaOH and HCl (see page 44 at DR0417-073). I made some of the buffers on that day, there was no time for making all the buffers. I measured the concentration of the IRM13 in Qubit found 0.714mg/ml. 12JAN18 Ι mixed the appropriate volumes of the acids and bases in the wells of a U-shaped plate. I added 100ul of buffer each time: CITRATE Acid (ul) Base (ul) 3.5         70.8 29.2 4 59 41 ...

03JAN18 After coming back from festive break, I tool my samples from the freezer and run 2 gels containing all of the AKTA IRM12 fractions (see gels on pages 41 and 42). The IRM12 protein is detected in F11,12,13 and 14 while there is also unspecific binding of other proteins. So I will be doing another AKTA (one that I dont have access to and I will do it with Anemmette). Using this AKTA I will do a size separation using a column. I made a 2lt of 50mM MES buffer A (recipe on page 116 DR 0817-073), 2lt of water and 1lt of Ethanol 20% and I degased them. I wash the AKTA with water, and leave it with buffer A to calibrate overnight. 04JAN18 I need to concentrate down the AKTA fractions where I detected the IRM12 protein (F11,12,13 and 14) to 2.5ml. The big AKTA size separation column can work with up to 5ml but it would be better to concentrate them to 2.5ml. This took the whole day spinning down at 4oC and topping up with more liquid. I kept the flowthrough. We run the big ...