11Jan-19Jan18
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:
Τhe above volumes are based on published recipes (see pages 42 and 45 in DR 0417-073). For the diagram of the plate see on page 46 at DR 1117-073.
I added 1ul of IRM13 protein (using a multichannel pipette). I incubated at 37oC for 5min. I quenched the reaction with 100ul of Na2CO3, I spun the plate for breaking bubbles and measured the absorbance at 405nm.
There is no activity to be detected for IRM13. It looks that the enzyme is no active at all.
I repeated the assay using B99 protein and 2 IRM13 produced in the past (stored at -80oC). See the plate diagram on page 47 at DR 1117-073.
The measured again the concentration using BCA method and it was found 76.4 ul/ml which is 10 times smaller than the concentration calculate yesterday with Qubit. I measured it again with Qubit and was found 0.61mg/ml which is very close to the concentration I measured yesterday. So it seems that Qubit measurement is closer to the reality. Also, looking at the gel on page the bands look like representing a concentration of close to 0.7mg/ml. I run a gel with IRM13 after dialysis and also I run the F27, F28, F29 and F30 smaples (kept SDS samples in the fridge) which I had combined for making the IRM13 pure protein. I would expect the F27-F30 bands combined to give the wide of the IRM13 pure protein band. It does not happen (see gel on page ).
15JAN2018
I sonicated some cells IRM13 this morning and extracted the CFE. I repeated the same activity assay as last Friday but this time I used the CFE along with the purified protein side by side. I also used some water as a control. I placed 100ul of phosphate buffer pH 7 50mM with 1.25mM PNPG and PNPX in the wells and added 25ul of the CFE. I also I added 10ul of the pure protein this time in case that the IRM13 protein we used the previous time is very little for revealing a reaction (1ul). I checked for activity at t=0, t=5 and t=30 min. I quenched the reaction with 50ul of Na2CO3. The activity of the CFE was so big that I had to dilute the sample in another well by 1/10. See the diagram of the plate on page 49 at DR1117-07.
16JAN18
There is a suggestion that IRM13 is thermo-sensitive and it does not like staying at 4oC for overnight. So the idea is the come early and try sonication-purification-dialysis-assay all in one day. I need to prepare the sonication buffer, degas the AKTA solutions, make dialysis buffer and make a plan
17JAN18
I came at 6 and started the sonication of IRM13 and some I17 (cells BL21(DE3) containg the Pet16b plasmid but lacking the IRM13 gene). Then produce the CFE (for both) and then AKTA(only for IRM13). I kept the I17 CFE in the freezer at -20oC for future use. I put the expected IRM13 AKTA fractions for dialysis (expected from the previous AKTA and I have noticed that IRM13 elutes at about 450mM Imidazole). In the meantime I checked all the AKTA fractions to detect my protein (see gels on pages ). There is at some other fractions more clear that the fractions I was dialysing. So I started again the dialysis using the best fractions. this costed 2 hours in terms of time but everything was in the fridge or in the ice. Once the dialysis fulfilled I repeated the pH assay with the dialysed protein. There are no results for activity. I also repeated the experiment using 10ul of my pure protein but again there are not results of activity. My protein seems inactive.
I checked its concentration with Qubit and it was found 0.371mg/ml
I also used some of the I17 CFE and repeated the activity assay (on PNPG and PNPX) for having the results as negative control. The activity values produced by the I17 are higher that those produced by the IRM13 pure protein activity. Maybe because the I17 cells have grown in TB overnight express medium which is a bit darker than the LB. In any case it was suggested to used just buffer without substrate and add protein as a control the next time.
18-19JAN18
There is an idea of the Imidazole or AKTA affecting the protein activity therefore there are the following suggestions:
1. Try some CFE activity assay but containing Imidazole in the buffer mix (same concentration as needed for the IRM13 to be eluted) at pH7.
2. Put some CFE in dialysis in case that there is a co-factor needed for the enzyme to activate and this co-factor will be dialysed at pH7
3. Collect the washes of the AKTA and check them for activity at pH7
4. Activity assay on AKTA fractions before dialysis pH7
These are very interesting ideas which are to be tested on Monday.
I set and overnight since I need more cells for starting on Monday.
I made the required sonication, AKTA degassing and dialysis buffers for Monday.
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 |
| 4.5 | 47 | 53 |
| 5 | 35 | 65 |
| 5.5 | 23.2 | 76.8 |
| PHOSPATE | ||
| Acid (ul) | Base (ul) | |
| 6 | 87.7 | 12.3 |
| 6.5 | 68.5 | 31.5 |
| 7 | 39 | 61 |
| 7.5 | 16 | 84 |
Τhe above volumes are based on published recipes (see pages 42 and 45 in DR 0417-073). For the diagram of the plate see on page 46 at DR 1117-073.
I added 1ul of IRM13 protein (using a multichannel pipette). I incubated at 37oC for 5min. I quenched the reaction with 100ul of Na2CO3, I spun the plate for breaking bubbles and measured the absorbance at 405nm.
There is no activity to be detected for IRM13. It looks that the enzyme is no active at all.
I repeated the assay using B99 protein and 2 IRM13 produced in the past (stored at -80oC). See the plate diagram on page 47 at DR 1117-073.
The measured again the concentration using BCA method and it was found 76.4 ul/ml which is 10 times smaller than the concentration calculate yesterday with Qubit. I measured it again with Qubit and was found 0.61mg/ml which is very close to the concentration I measured yesterday. So it seems that Qubit measurement is closer to the reality. Also, looking at the gel on page the bands look like representing a concentration of close to 0.7mg/ml. I run a gel with IRM13 after dialysis and also I run the F27, F28, F29 and F30 smaples (kept SDS samples in the fridge) which I had combined for making the IRM13 pure protein. I would expect the F27-F30 bands combined to give the wide of the IRM13 pure protein band. It does not happen (see gel on page ).
15JAN2018
I sonicated some cells IRM13 this morning and extracted the CFE. I repeated the same activity assay as last Friday but this time I used the CFE along with the purified protein side by side. I also used some water as a control. I placed 100ul of phosphate buffer pH 7 50mM with 1.25mM PNPG and PNPX in the wells and added 25ul of the CFE. I also I added 10ul of the pure protein this time in case that the IRM13 protein we used the previous time is very little for revealing a reaction (1ul). I checked for activity at t=0, t=5 and t=30 min. I quenched the reaction with 50ul of Na2CO3. The activity of the CFE was so big that I had to dilute the sample in another well by 1/10. See the diagram of the plate on page 49 at DR1117-07.
16JAN18
There is a suggestion that IRM13 is thermo-sensitive and it does not like staying at 4oC for overnight. So the idea is the come early and try sonication-purification-dialysis-assay all in one day. I need to prepare the sonication buffer, degas the AKTA solutions, make dialysis buffer and make a plan
17JAN18
I came at 6 and started the sonication of IRM13 and some I17 (cells BL21(DE3) containg the Pet16b plasmid but lacking the IRM13 gene). Then produce the CFE (for both) and then AKTA(only for IRM13). I kept the I17 CFE in the freezer at -20oC for future use. I put the expected IRM13 AKTA fractions for dialysis (expected from the previous AKTA and I have noticed that IRM13 elutes at about 450mM Imidazole). In the meantime I checked all the AKTA fractions to detect my protein (see gels on pages ). There is at some other fractions more clear that the fractions I was dialysing. So I started again the dialysis using the best fractions. this costed 2 hours in terms of time but everything was in the fridge or in the ice. Once the dialysis fulfilled I repeated the pH assay with the dialysed protein. There are no results for activity. I also repeated the experiment using 10ul of my pure protein but again there are not results of activity. My protein seems inactive.
I checked its concentration with Qubit and it was found 0.371mg/ml
I also used some of the I17 CFE and repeated the activity assay (on PNPG and PNPX) for having the results as negative control. The activity values produced by the I17 are higher that those produced by the IRM13 pure protein activity. Maybe because the I17 cells have grown in TB overnight express medium which is a bit darker than the LB. In any case it was suggested to used just buffer without substrate and add protein as a control the next time.
18-19JAN18
There is an idea of the Imidazole or AKTA affecting the protein activity therefore there are the following suggestions:
1. Try some CFE activity assay but containing Imidazole in the buffer mix (same concentration as needed for the IRM13 to be eluted) at pH7.
2. Put some CFE in dialysis in case that there is a co-factor needed for the enzyme to activate and this co-factor will be dialysed at pH7
3. Collect the washes of the AKTA and check them for activity at pH7
4. Activity assay on AKTA fractions before dialysis pH7
These are very interesting ideas which are to be tested on Monday.
I set and overnight since I need more cells for starting on Monday.
I made the required sonication, AKTA degassing and dialysis buffers for Monday.
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