08NOV17 In vitro protein expression for IRM 1,2,3,4,5 / Purification of IRM12
08NOV17
-IRM12 PURIFICATION
The gel staining overnight showed that there must be some protein in the 50mM and 150mM Imidazole lanes (see page for the gel).
I used 10kDa filter cut off tubes (pink lid) to filter down my protein. I wash the filter by fill it up with water and spin it down at 4000rpm at 4oC for 10min twice.
I need to concentrate my protein down to 500μl so I am filling up the filter in the tube and spin it down to 4000rpm for 20 minutes at 4oC. Each time the run finishes, I collect the flow through, fill the filter up with more of the buffer+protein solution, shake it again to prevent any blockage on the filter and spin it again. This procedure is slow because either the protein blocks the filter or the solution contains other substances which block the filter. In any case I need to help the filter goes by either shaking up and down the tube or help pipetting up and down close to the filter. I put the tube in the centrifuge so that both filters are interacting with the solution.
I started with the less concentrated solution in terms of protein (150mM Imidazole). I then add the 50mM imidazole-protein buffer. Once I finished and have 500μl of protein in the filter I will start the washes (tomorrow since the time run off).
I need to wash with a buffer I will use for assays next. Lets say a neutral buffer. The washes will help the Imidazole to dilute and get in the flow through. I need about 3-4 washes with buffer. The accepted concentration of Imidazole in my buffer-protein solution is less/equal than/to 5. I will use sodium phosphate buffer at 7.0 pH.
-In Vitro protein expression NEB kit - EVERYTHING NEEDS TO BE VERY CAREFULLY CLEAN. HANDS, GLOVES AND BENCHES (TO AVOID PROTEASES IN THE MIX)
First I need to calculate the concentration of my DNA. I used Qubit. There are 2 standards for measuring DNA in the fridge. I made a master mix of 199μl of the DNA buffer and 1μl of DNA stain for each sample. Then I added 190μl of this diluted stain for the standards and 10μl of the standards into Qubit tubes and 199μl of the diluted stain and 1μl of my sample in Qubit tubes. I measured the concentration (High sensitivity) in ng/μl:
IRM1: 66.6
IRM2: 81.8
IRM3: 57.6
IRM4: 17.7
IRM5: 53.4
B99 (as positive control): 95.4
I followed the instruction in the kit. I prepared 6 PCR tubes for IRM1,2,3,4,5 and B99. I added 10μl of Solution A in the tubes, then 7.5 μl of Solution B. Then I calculated the amount of DNA I need to add. In the instruction it says they recommend to use 250ng of DNA in the 25μl of reaction. So by using the C1V1 = C2V2 and the above DNA concentrations we find that we need to add the following volume of DNA:
IRM1: 3.75μl
IRM2: 3.06 μl
IRM3: 4.34μl
IRM4: 14.12μl
IRM5: 4.68 μl
B99: 2.62μl
So we need to make up a 25μl reaction with water. I use autocleaved water. So 17.5μl of the solution A and B + the volume of DNA and then I just add autocleaved water (in the storage room aliquoted in white-blue lid small tubes). For the IRM4 I didnt add any water just the DNA to make it up to 25μl.
I incubate in a PCR machine for 4 hours at 37oC. I keep the produced proteins in their tubes in 4oC (fridge).
-TAPE STATION PREP
I followed the instructions in the Agilent P200 ScreenTape System Quick Guide. The reagents are in the -20oC in a small black box in the drawer Annete in the stock room freezer.
I make a master mix of diluted labeling dye 1:5 for each sample. I have 6 samples but also I have a ladder and a blank so all together 8 samples. So I will make diluted stain for lets say 10 samples. So I added 16 μl of labeling buffer and 4μl of labeling stain.
I placed 2 μl of the diluted stain in the specific tubes for the TapeStation machine. I added 2μl of my protein or stain and just water for the blank. Place the lids and vortex. Heat up at 37oC for 7min in the PCR machine.
Centrifuge for removing the condensation.
Add 4μl of P200 reducing sample buffer to each tube and ladder, vortex and heat up to 37oC for 5min.
Remove condensation with centrifugation.
Add 2μl of P200 marker to each sample and to the ladder, vortex and centrifuge to have the solutions at the bottom of the tubes.
Now I am ready to use the TapeStation machine. I load the tubes in the spaces at the left of the machine and take off their lids. I also fill up the middle spaces of pipetting tips. I place a screentape to the right side of the machine. I can find the screentape in the silver fridge in storage room in a white box with white lid called RNA and screentape.
I close the machine and control it from the PC by the machine. The program controlling the machine is called 2200 TapeStation Controller.
I select the tubes I have used on the program, name the samples and run it. At the end the results can be exported in pdf or stored in the computer (for results see page ).
Tomorrow I will run the PNPG and PNPX assays using the produced protein from the in vitro kit. I will need 99μl of buffer-PNPG/PNPX and 1μl of my protein sample. I will test the 5min and 30min incubation time at 37oC and quench the reaction with 50 μl of Na2CO3. I will measure the color change.
-IRM12 PURIFICATION
The gel staining overnight showed that there must be some protein in the 50mM and 150mM Imidazole lanes (see page for the gel).
I used 10kDa filter cut off tubes (pink lid) to filter down my protein. I wash the filter by fill it up with water and spin it down at 4000rpm at 4oC for 10min twice.
I need to concentrate my protein down to 500μl so I am filling up the filter in the tube and spin it down to 4000rpm for 20 minutes at 4oC. Each time the run finishes, I collect the flow through, fill the filter up with more of the buffer+protein solution, shake it again to prevent any blockage on the filter and spin it again. This procedure is slow because either the protein blocks the filter or the solution contains other substances which block the filter. In any case I need to help the filter goes by either shaking up and down the tube or help pipetting up and down close to the filter. I put the tube in the centrifuge so that both filters are interacting with the solution.
I started with the less concentrated solution in terms of protein (150mM Imidazole). I then add the 50mM imidazole-protein buffer. Once I finished and have 500μl of protein in the filter I will start the washes (tomorrow since the time run off).
I need to wash with a buffer I will use for assays next. Lets say a neutral buffer. The washes will help the Imidazole to dilute and get in the flow through. I need about 3-4 washes with buffer. The accepted concentration of Imidazole in my buffer-protein solution is less/equal than/to 5. I will use sodium phosphate buffer at 7.0 pH.
-In Vitro protein expression NEB kit - EVERYTHING NEEDS TO BE VERY CAREFULLY CLEAN. HANDS, GLOVES AND BENCHES (TO AVOID PROTEASES IN THE MIX)
First I need to calculate the concentration of my DNA. I used Qubit. There are 2 standards for measuring DNA in the fridge. I made a master mix of 199μl of the DNA buffer and 1μl of DNA stain for each sample. Then I added 190μl of this diluted stain for the standards and 10μl of the standards into Qubit tubes and 199μl of the diluted stain and 1μl of my sample in Qubit tubes. I measured the concentration (High sensitivity) in ng/μl:
IRM1: 66.6
IRM2: 81.8
IRM3: 57.6
IRM4: 17.7
IRM5: 53.4
B99 (as positive control): 95.4
I followed the instruction in the kit. I prepared 6 PCR tubes for IRM1,2,3,4,5 and B99. I added 10μl of Solution A in the tubes, then 7.5 μl of Solution B. Then I calculated the amount of DNA I need to add. In the instruction it says they recommend to use 250ng of DNA in the 25μl of reaction. So by using the C1V1 = C2V2 and the above DNA concentrations we find that we need to add the following volume of DNA:
IRM1: 3.75μl
IRM2: 3.06 μl
IRM3: 4.34μl
IRM4: 14.12μl
IRM5: 4.68 μl
B99: 2.62μl
So we need to make up a 25μl reaction with water. I use autocleaved water. So 17.5μl of the solution A and B + the volume of DNA and then I just add autocleaved water (in the storage room aliquoted in white-blue lid small tubes). For the IRM4 I didnt add any water just the DNA to make it up to 25μl.
I incubate in a PCR machine for 4 hours at 37oC. I keep the produced proteins in their tubes in 4oC (fridge).
-TAPE STATION PREP
I followed the instructions in the Agilent P200 ScreenTape System Quick Guide. The reagents are in the -20oC in a small black box in the drawer Annete in the stock room freezer.
I make a master mix of diluted labeling dye 1:5 for each sample. I have 6 samples but also I have a ladder and a blank so all together 8 samples. So I will make diluted stain for lets say 10 samples. So I added 16 μl of labeling buffer and 4μl of labeling stain.
I placed 2 μl of the diluted stain in the specific tubes for the TapeStation machine. I added 2μl of my protein or stain and just water for the blank. Place the lids and vortex. Heat up at 37oC for 7min in the PCR machine.
Centrifuge for removing the condensation.
Add 4μl of P200 reducing sample buffer to each tube and ladder, vortex and heat up to 37oC for 5min.
Remove condensation with centrifugation.
Add 2μl of P200 marker to each sample and to the ladder, vortex and centrifuge to have the solutions at the bottom of the tubes.
Now I am ready to use the TapeStation machine. I load the tubes in the spaces at the left of the machine and take off their lids. I also fill up the middle spaces of pipetting tips. I place a screentape to the right side of the machine. I can find the screentape in the silver fridge in storage room in a white box with white lid called RNA and screentape.
I close the machine and control it from the PC by the machine. The program controlling the machine is called 2200 TapeStation Controller.
I select the tubes I have used on the program, name the samples and run it. At the end the results can be exported in pdf or stored in the computer (for results see page ).
Tomorrow I will run the PNPG and PNPX assays using the produced protein from the in vitro kit. I will need 99μl of buffer-PNPG/PNPX and 1μl of my protein sample. I will test the 5min and 30min incubation time at 37oC and quench the reaction with 50 μl of Na2CO3. I will measure the color change.
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