27NOV-07DEC AKTA for IRM12, in vitro protein synthesis
27NOV17
I prepared 1lt of water, 1/2 lt of buffer A, 1/2 lt buffer B for IRM12 and 1/2 lt of Ethanol 20%. I need to degass them before AKTA.
I took a fennel (storage room at the back in a plastic box), a cylinder 1lt and 4 stirring stones. I put the water into the cylinder. Place the stirring stone in the empty water bottle and place it on the stirring plate. I adjusted the fennel onto the bottle and strewed it and then I connected the degassing tube to the fennel. I turn the degassing machine on and pour some of the water very gently into the fennel. I want the filter to be soaked first and then I put the rest of the water. I place the lid and turn the stirring plate on. The degassing has been finished once there no bubbles on the stirring stone.
The degased buffers can be kept for a day degased so I can run the AKTA tomorrow morning
28NOV17
I run the AKTA using the degased buffers for purifying the IRM12 and collected all the 30 fractions produced. I also saved the session in the computer. I also degased the buffers for the IRM13
29NOV17
I run all the frctions of IRM12 on the gel (see gels on pages ) but I could not place my protein in any of the fractions. I run the AKTA for the IRM13
30NOV17 and 1DEC17
I run the fractions where I saw a pick in the AKTA program on a gel (see gel on page ). The protein was detected on the second cycle of fraction (with an N by the number of the fraction) in F1N and F2N so I decided to run the F30, F3N and F4N on a gel (see gels on pages ). There is protein in F30, F3N and F4N but there are other unspecific bound proteins as well so I decided to combine the F1N and F2N and wash them with buffer A and centrifuge them down to 3ml (from the 6ml each fraction was). I didnt do dialysis because of the coming weekend. I washed with buffer A 5 times and concentrate down to 3ml. I stored the protein at -20oC for over the weekend.
4DEC17
I run the pure IRM13 protein on a gel (see the gel on page ). It is not very intense and its fainter that the bands on the gel on the AKTA gel 30NOV17. This means that maybe my protein has been lost somewhere. I run the washes and the flowthrough from when I was washing the protein and concentrating it down on a gel to see whether my protein has been washed off (unlikely because the filter 10kDa cut off allows only the smaller than 10kDa molecules to be washed off).
5DEC17
There was no protein detected on the gel (see gel on page ). I need to estimate the concentration of the IRM13. I did a BCA assay and the result showed that the concentration of the protein is 83.8μg/ml which explains why the band is so faint. But the bands of the AKTA fractions indicate higher concentration. So maybe my protein is not stable and it got degrated while keeping it int he fridge and freezer. What I need to do is some scaling up for producing more biomass, purify it and go straight to the assays.
6DEC17
Looking at PNPG and PNPX assay with the cell-free NEB kit on page DR1117-073 and the Tapestation gels, we are thinking that maybe the problem with the produced proteins was that I havent used an RNAase inhibitor. Another point that Leo said is that the B99 was produced with miniprep while we dont know how the Genewiz produced the DNA sequences.
In any case we decided to order an RNAase inhibitor and repeat the experiment for a positive and blank sample with and without RNAase inhibitor (that I have cell-free kit to produce blank and positive sample) and if there is a need to order a new kit afterwards and repeat the protein production for all of them.
I run a western blot today for all of the IRM samples (produced by the NEB in-vitro protein production), the DNA template positive of the kit, the B99 positive to my experiment. Since it needed only 2.5μl of sample to run a gel (it is written in the kit instructions, I had so little amount left) I run a gel and then a western.
7DEC17
I stained the western
*discrepancy from the usual staining procedure*
For staining the western membrane I added 10ml of stable peroxide 1X found in the silver fridge and 100μl of DAB found in a plastic box in the third drawer of the freezer in the storage room).
I stain again for 5 min. The westerns (pages ) show that it might be a problem possibly due to the lack of RNAase inhibitor in the in-vitro protein synthesis reaction.
I received the RNAase inhibitor today and I tried to synthesise the DNA template positive for the kit, the B99 positive for my experiment and the blank with RNAase inhibitor. I incubated the samples for 4 hours. I kept the samples in the freezer
I prepared 1lt of water, 1/2 lt of buffer A, 1/2 lt buffer B for IRM12 and 1/2 lt of Ethanol 20%. I need to degass them before AKTA.
I took a fennel (storage room at the back in a plastic box), a cylinder 1lt and 4 stirring stones. I put the water into the cylinder. Place the stirring stone in the empty water bottle and place it on the stirring plate. I adjusted the fennel onto the bottle and strewed it and then I connected the degassing tube to the fennel. I turn the degassing machine on and pour some of the water very gently into the fennel. I want the filter to be soaked first and then I put the rest of the water. I place the lid and turn the stirring plate on. The degassing has been finished once there no bubbles on the stirring stone.
The degased buffers can be kept for a day degased so I can run the AKTA tomorrow morning
28NOV17
I run the AKTA using the degased buffers for purifying the IRM12 and collected all the 30 fractions produced. I also saved the session in the computer. I also degased the buffers for the IRM13
29NOV17
I run all the frctions of IRM12 on the gel (see gels on pages ) but I could not place my protein in any of the fractions. I run the AKTA for the IRM13
30NOV17 and 1DEC17
I run the fractions where I saw a pick in the AKTA program on a gel (see gel on page ). The protein was detected on the second cycle of fraction (with an N by the number of the fraction) in F1N and F2N so I decided to run the F30, F3N and F4N on a gel (see gels on pages ). There is protein in F30, F3N and F4N but there are other unspecific bound proteins as well so I decided to combine the F1N and F2N and wash them with buffer A and centrifuge them down to 3ml (from the 6ml each fraction was). I didnt do dialysis because of the coming weekend. I washed with buffer A 5 times and concentrate down to 3ml. I stored the protein at -20oC for over the weekend.
4DEC17
I run the pure IRM13 protein on a gel (see the gel on page ). It is not very intense and its fainter that the bands on the gel on the AKTA gel 30NOV17. This means that maybe my protein has been lost somewhere. I run the washes and the flowthrough from when I was washing the protein and concentrating it down on a gel to see whether my protein has been washed off (unlikely because the filter 10kDa cut off allows only the smaller than 10kDa molecules to be washed off).
5DEC17
There was no protein detected on the gel (see gel on page ). I need to estimate the concentration of the IRM13. I did a BCA assay and the result showed that the concentration of the protein is 83.8μg/ml which explains why the band is so faint. But the bands of the AKTA fractions indicate higher concentration. So maybe my protein is not stable and it got degrated while keeping it int he fridge and freezer. What I need to do is some scaling up for producing more biomass, purify it and go straight to the assays.
6DEC17
Looking at PNPG and PNPX assay with the cell-free NEB kit on page DR1117-073 and the Tapestation gels, we are thinking that maybe the problem with the produced proteins was that I havent used an RNAase inhibitor. Another point that Leo said is that the B99 was produced with miniprep while we dont know how the Genewiz produced the DNA sequences.
In any case we decided to order an RNAase inhibitor and repeat the experiment for a positive and blank sample with and without RNAase inhibitor (that I have cell-free kit to produce blank and positive sample) and if there is a need to order a new kit afterwards and repeat the protein production for all of them.
I run a western blot today for all of the IRM samples (produced by the NEB in-vitro protein production), the DNA template positive of the kit, the B99 positive to my experiment. Since it needed only 2.5μl of sample to run a gel (it is written in the kit instructions, I had so little amount left) I run a gel and then a western.
7DEC17
I stained the western
*discrepancy from the usual staining procedure*
For staining the western membrane I added 10ml of stable peroxide 1X found in the silver fridge and 100μl of DAB found in a plastic box in the third drawer of the freezer in the storage room).
I stain again for 5 min. The westerns (pages ) show that it might be a problem possibly due to the lack of RNAase inhibitor in the in-vitro protein synthesis reaction.
I received the RNAase inhibitor today and I tried to synthesise the DNA template positive for the kit, the B99 positive for my experiment and the blank with RNAase inhibitor. I incubated the samples for 4 hours. I kept the samples in the freezer
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