15NOV17 purification small scale IRM13
14NOV17
After searching in the yak paper for finding how much protein has been used in the assays (so I know how much protein I need myself), I found that they have not stated how much protein they use in any of their experiments.
According to the gel I run using the pure IRM12 protein produced, I need to use closer Imidazole concentration steps so that the contaminant proteins will eluted in the inbetween steps before my protein will elute.
I was about to repeat a small scale purification of IRM13 but I realised I dont have any CHAPS (detergent I use in the sonication buffer). Instead I have Triton (which can substitute CHAPS) according to DR 0817-073 page 116. So I repeated the sonication and purification procedure using Triton in the sonication buffer. I did a purification of IRM12 and IRM13 with Triton instead of CHAPS.
I sonicate the IRM12 and IRM13. The sonication mix got very foamy because of the Triton used. The sonication bubbles (need to be formed so that the sonicate works) were not visible because of the foam. I was not sure if the sonication works. I continue to see at the end of the experiment.
I spun down the lysate at 10.000rpm at 4oC for 30min, I took samples of the CFE and the insoluble fraction, I put the pre-washed and equilibrated resin and left it 2 hours for incubation on the rocker in the cold room. I purified the proteins using the following Imidazole concentration steps:
IRM12:
50mM MES, NaCl and 20mM Imidazole
50mM MES, NaCl and 50mM Imidazole
50mM MES, NaCl and 70mM Imidazole
50mM MES, NaCl and 100mM Imidazole
50mM MES, NaCl and 120mM Imidazole
IRM13:
50mM TRIS, NaCl and 20mM Imidazole
50mM TRIS, NaCl and 50mM Imidazole
50mM TRIS, NaCl and 70mM Imidazole
50mM TRIS, NaCl and 100mM Imidazole
50mM TRIS, NaCl and 120mM Imidazole
50mM TRIS, NaCl and 250mM Imidazole
50mM TRIS, NaCl and 350mM Imidazole
I made a mix of the standard A and standard B buffers which also have a different NaCl concentration but I can calculate the concentration using the method in DR 1117-073 page 3.
I purified the IRM13 since I didnt have enough buffer for the IRM12 and it was already late.
After searching in the yak paper for finding how much protein has been used in the assays (so I know how much protein I need myself), I found that they have not stated how much protein they use in any of their experiments.
According to the gel I run using the pure IRM12 protein produced, I need to use closer Imidazole concentration steps so that the contaminant proteins will eluted in the inbetween steps before my protein will elute.
I was about to repeat a small scale purification of IRM13 but I realised I dont have any CHAPS (detergent I use in the sonication buffer). Instead I have Triton (which can substitute CHAPS) according to DR 0817-073 page 116. So I repeated the sonication and purification procedure using Triton in the sonication buffer. I did a purification of IRM12 and IRM13 with Triton instead of CHAPS.
I sonicate the IRM12 and IRM13. The sonication mix got very foamy because of the Triton used. The sonication bubbles (need to be formed so that the sonicate works) were not visible because of the foam. I was not sure if the sonication works. I continue to see at the end of the experiment.
I spun down the lysate at 10.000rpm at 4oC for 30min, I took samples of the CFE and the insoluble fraction, I put the pre-washed and equilibrated resin and left it 2 hours for incubation on the rocker in the cold room. I purified the proteins using the following Imidazole concentration steps:
IRM12:
50mM MES, NaCl and 20mM Imidazole
50mM MES, NaCl and 50mM Imidazole
50mM MES, NaCl and 70mM Imidazole
50mM MES, NaCl and 100mM Imidazole
50mM MES, NaCl and 120mM Imidazole
IRM13:
50mM TRIS, NaCl and 20mM Imidazole
50mM TRIS, NaCl and 50mM Imidazole
50mM TRIS, NaCl and 70mM Imidazole
50mM TRIS, NaCl and 100mM Imidazole
50mM TRIS, NaCl and 120mM Imidazole
50mM TRIS, NaCl and 250mM Imidazole
50mM TRIS, NaCl and 350mM Imidazole
I made a mix of the standard A and standard B buffers which also have a different NaCl concentration but I can calculate the concentration using the method in DR 1117-073 page 3.
I purified the IRM13 since I didnt have enough buffer for the IRM12 and it was already late.
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