Ingenza

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 ...

18-22DEC17 I made a new IRM12 E.coli culture and sonicate it using the DR 0817-073 page 116 recipe only with 1% glycerol always. I took the lysate and run an AKTA. The AKTA computer was not starting up so I did it manually using only the machine. The data were not stored and therefore I am not able to see at the details of the AKTA but I did take the fractions out. I froze everything at -20oC to stay over the Christmas period and I will run a gel containing all the fractions for detecting the protein after the company reopen again.