09NOV17
IRM12 PURIFICATION AND CONCENTRATION MEASURMENT
I purify the IRM12 by spin it down and wash it with 15ml of sodium phosphate pH 7 . At end I had 500μl of pure protein (with glycerol and maybe other contaminants).
I measured the concentration of the protein in Qubit and it was 2440ng/μl (I needed a lot of dilutions to be able to measure it, it seemed the concentration was very high).
I measure also the IRM12 absorbance in 280nm in the high sensitivity spectrophotometre. I diluted the protein first 1:30, then 1:4 and then 1:1 with the buffer I used to wash it (sodium phosphate pH 7). I blank with sodium phosphate pH 7 buffer and measured the absorbance at 280nm. So after all these dilutions the absorbance number was 0,463
So the absorbance of the original protein solution would be :
Afinal solution = Dilution factor x Aoriginal
Afinal solution = 1/30 x 1/4 x 1/2 x 0.463
Aoriginal = 240 Afinal solution
Aoriginal = 240 x 0463
Aoriginal = 111.12
A = E x p x c
c = A/E x p
c = 111.12/109725 (extinction coefficient of the protein found in expasy page 110 of DR 0817-073) x 1
c = 1.01 x 10E-03 M
c = 1 mM
However, I am not sure whether other proteins exist in the solution and maybe the absorbance at 280nm is due to other aromatic rings in other proteins that absorb in this wavelength.
I need to run a gel to see whether other proteins exist. I prepared a sample to run in at the beginning of next week.
I also stored my protein with 20% glycerol at -80oC.
-PNPG and PNPX EXPERIMENT
I prepared 2 plates using the protein of IRM1,2,3,4,5,B99 I have produced using the invitro kit yesterday. The plates look like:
IRM1 PNPG IRM2 PNPG (etc with IRM3, 4, 5 and B99 ie. 6 columns)
-
IRM1 PNPG IRM2 PNPG
IRM1 PNPG IRM2 PNPG
IRM1 PNPX IRM2PNPX
IRM1 PNPX IRM2 PNPX
IRM1 PNPX IRM2 PNPX
I prepared 2 plates one for the t=5min and one for the t=30min
I put 1μl of the protein and then 99μl of the buffer sodium phosphate pH7 + PNPG/PNPX. After the incubation time, I quenched the reaction with 50μl of Na2CO3. I measured the absorbance at 405nm and made graphs.
IRM12 PURIFICATION AND CONCENTRATION MEASURMENT
I purify the IRM12 by spin it down and wash it with 15ml of sodium phosphate pH 7 . At end I had 500μl of pure protein (with glycerol and maybe other contaminants).
I measured the concentration of the protein in Qubit and it was 2440ng/μl (I needed a lot of dilutions to be able to measure it, it seemed the concentration was very high).
I measure also the IRM12 absorbance in 280nm in the high sensitivity spectrophotometre. I diluted the protein first 1:30, then 1:4 and then 1:1 with the buffer I used to wash it (sodium phosphate pH 7). I blank with sodium phosphate pH 7 buffer and measured the absorbance at 280nm. So after all these dilutions the absorbance number was 0,463
So the absorbance of the original protein solution would be :
Afinal solution = Dilution factor x Aoriginal
Afinal solution = 1/30 x 1/4 x 1/2 x 0.463
Aoriginal = 240 Afinal solution
Aoriginal = 240 x 0463
Aoriginal = 111.12
A = E x p x c
c = A/E x p
c = 111.12/109725 (extinction coefficient of the protein found in expasy page 110 of DR 0817-073) x 1
c = 1.01 x 10E-03 M
c = 1 mM
However, I am not sure whether other proteins exist in the solution and maybe the absorbance at 280nm is due to other aromatic rings in other proteins that absorb in this wavelength.
I need to run a gel to see whether other proteins exist. I prepared a sample to run in at the beginning of next week.
I also stored my protein with 20% glycerol at -80oC.
-PNPG and PNPX EXPERIMENT
I prepared 2 plates using the protein of IRM1,2,3,4,5,B99 I have produced using the invitro kit yesterday. The plates look like:
IRM1 PNPG IRM2 PNPG (etc with IRM3, 4, 5 and B99 ie. 6 columns)
-
IRM1 PNPG IRM2 PNPG
IRM1 PNPG IRM2 PNPG
IRM1 PNPX IRM2PNPX
IRM1 PNPX IRM2 PNPX
IRM1 PNPX IRM2 PNPX
I prepared 2 plates one for the t=5min and one for the t=30min
I put 1μl of the protein and then 99μl of the buffer sodium phosphate pH7 + PNPG/PNPX. After the incubation time, I quenched the reaction with 50μl of Na2CO3. I measured the absorbance at 405nm and made graphs.
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