22JAN-26JAN
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 inhibits the enzyme activity (or even denatures it however I didnt see any precipitation). I repeated the experiment with the CFE activity + Imidazole but this time I made a Imidazole concentration gradient. I tested the CFE for concentration at 500mM, 50mM, 5mM, 0.5mM and 0mM Imidazole. Again 100ul of buffer+substrate+imidazole conc. and then 25ul of the CFE with the multi-pipette and incubation for 5 min at 37oC and quench with 50ul Na2CO3 and measure at 405nm. See the diagram of the plate on page
I had to dilute the solution in the wells for the Imidazole concentration of 5mM, 0.5mM and 0mM because the absorbance was very high. It seems that even 50mM Imidazole inhibits a lot the enzymes activity. The idea was to start checking for that broad range of Imidazole concentration and narrow down the Imidazole concentration for checking activity. The weird thing is that dialysis theoritically works as reduces the concentration of Imidazole in the snakebag down to 1.2mM (C1V1=C2V2 where C1 is the conc of Imidazole in the bag, V1 is the volume of the sample in the bag and V2 is the volume of the buffer in which I dialysed+the sample volume). So C2=C1V1/V2 => C2=225mM x 10ml/2000+10 => C2=2250/2010mM => C2=1.12mM left Imidazole concentration in the bag after dialysis.
I should have activity then using the dialysed protein if the only problem was the Imidazole concentration since the activity Imidazole concentration range proved that in 5mM Imidazole I have good activity. There seem to be something else.
So I dialysed overnight 10ml of sample while I left the some sample in the fridge not dialysed. this way Im testing whether there is a co-factor required for my enzyme to act (and this co-factor is dialysed and passes out of the snakeskin bag) or my enxyme is very thermo-unstable and it doesnt like being kept in the fridge for long.
23JAN
I tested for activity for dialysed and non-dialysed CFE. I placed 100ul of phosphate buffer pH7 + substrate in the wells and put 25ul of dialysed CFE and non dialysed CFE. The control is buffer phosphate pH7 without substrate. I left them at 37oC for 5 min and quenched the reactions and measured at 405nm. It seems that there is some activity in the non-dialysed CFE but the dialysed CFE is totally inactive (same valuea s the controls). See the diagram of the plate on page
There must be a required co-factor for the activity reaction. I will use the ions they used in the yak paper in the concentrations they used and checked for activity in the dialysed CFE. My control should be the non-dialysed protein + substrate without ions. I will measure at 5 abd 30min.
I made some phosphate buffer pH7 with substrate and 5mM of MnCl2, MgCl2, CaCl2 and ZnCl2 to check whether these ions enhance the enzyme activity. I placed 100ul of the buffer+substrate+ions in the wells and used the non-dialysed CFE as a control (25ul). I also added some dialysed CFE without ions only substrate.See the diagram of the plate on page
I saw better activity of the CFE non-dialysed compared to the one from the morning's measurement possibly due to fresh buffer+fresh substrate I made in the afternoon. I need to repet the experiment as due to very low volumes the iosn concentration was not accurate and the absorbance values at 405nm are not reliable.
I have to test more ions and see in BRENDA database and test many ions.
24JAN18
I prepared phosphate buffer pH7 containing 1.25mM of PNPG and 50mM of some salts (so I can test for the ions effect to the dialysed IRM13 CFE I have kept in the fridge for 2 days now). The salts are KCl, CaCl2, MnCl2, MgCl2, FeCl2, and ZnCl2 I added 100ul of the buffer + ions in the wells of the 96 well plate, I also added 100ul of buffer pH7 + substrate without ions and tested the non dialysed CFE and the dialysed CFE. I measured at t=5 and t=30 at 405nm. The non-dialysed CFE is still active while there is some effect in the presence of some ions. The t0 measurement could not be saved (or lost while saving. The data below are from the t30 measurement.
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 inhibits the enzyme activity (or even denatures it however I didnt see any precipitation). I repeated the experiment with the CFE activity + Imidazole but this time I made a Imidazole concentration gradient. I tested the CFE for concentration at 500mM, 50mM, 5mM, 0.5mM and 0mM Imidazole. Again 100ul of buffer+substrate+imidazole conc. and then 25ul of the CFE with the multi-pipette and incubation for 5 min at 37oC and quench with 50ul Na2CO3 and measure at 405nm. See the diagram of the plate on page
I had to dilute the solution in the wells for the Imidazole concentration of 5mM, 0.5mM and 0mM because the absorbance was very high. It seems that even 50mM Imidazole inhibits a lot the enzymes activity. The idea was to start checking for that broad range of Imidazole concentration and narrow down the Imidazole concentration for checking activity. The weird thing is that dialysis theoritically works as reduces the concentration of Imidazole in the snakebag down to 1.2mM (C1V1=C2V2 where C1 is the conc of Imidazole in the bag, V1 is the volume of the sample in the bag and V2 is the volume of the buffer in which I dialysed+the sample volume). So C2=C1V1/V2 => C2=225mM x 10ml/2000+10 => C2=2250/2010mM => C2=1.12mM left Imidazole concentration in the bag after dialysis.
I should have activity then using the dialysed protein if the only problem was the Imidazole concentration since the activity Imidazole concentration range proved that in 5mM Imidazole I have good activity. There seem to be something else.
So I dialysed overnight 10ml of sample while I left the some sample in the fridge not dialysed. this way Im testing whether there is a co-factor required for my enzyme to act (and this co-factor is dialysed and passes out of the snakeskin bag) or my enxyme is very thermo-unstable and it doesnt like being kept in the fridge for long.
23JAN
I tested for activity for dialysed and non-dialysed CFE. I placed 100ul of phosphate buffer pH7 + substrate in the wells and put 25ul of dialysed CFE and non dialysed CFE. The control is buffer phosphate pH7 without substrate. I left them at 37oC for 5 min and quenched the reactions and measured at 405nm. It seems that there is some activity in the non-dialysed CFE but the dialysed CFE is totally inactive (same valuea s the controls). See the diagram of the plate on page
There must be a required co-factor for the activity reaction. I will use the ions they used in the yak paper in the concentrations they used and checked for activity in the dialysed CFE. My control should be the non-dialysed protein + substrate without ions. I will measure at 5 abd 30min.
I made some phosphate buffer pH7 with substrate and 5mM of MnCl2, MgCl2, CaCl2 and ZnCl2 to check whether these ions enhance the enzyme activity. I placed 100ul of the buffer+substrate+ions in the wells and used the non-dialysed CFE as a control (25ul). I also added some dialysed CFE without ions only substrate.See the diagram of the plate on page
I saw better activity of the CFE non-dialysed compared to the one from the morning's measurement possibly due to fresh buffer+fresh substrate I made in the afternoon. I need to repet the experiment as due to very low volumes the iosn concentration was not accurate and the absorbance values at 405nm are not reliable.
I have to test more ions and see in BRENDA database and test many ions.
24JAN18
I prepared phosphate buffer pH7 containing 1.25mM of PNPG and 50mM of some salts (so I can test for the ions effect to the dialysed IRM13 CFE I have kept in the fridge for 2 days now). The salts are KCl, CaCl2, MnCl2, MgCl2, FeCl2, and ZnCl2 I added 100ul of the buffer + ions in the wells of the 96 well plate, I also added 100ul of buffer pH7 + substrate without ions and tested the non dialysed CFE and the dialysed CFE. I measured at t=5 and t=30 at 405nm. The non-dialysed CFE is still active while there is some effect in the presence of some ions. The t0 measurement could not be saved (or lost while saving. The data below are from the t30 measurement.
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