03-10JAN18 big AKTA size separation column for discarding unspecific binding
03JAN18
After coming back from festive break, I tool my samples from the freezer and run 2 gels containing all of the AKTA IRM12 fractions (see gels on pages 41 and 42). The IRM12 protein is detected in F11,12,13 and 14 while there is also unspecific binding of other proteins. So I will be doing another AKTA (one that I dont have access to and I will do it with Anemmette). Using this AKTA I will do a size separation using a column.
I made a 2lt of 50mM MES buffer A (recipe on page 116 DR 0817-073), 2lt of water and 1lt of Ethanol 20% and I degased them.
I wash the AKTA with water, and leave it with buffer A to calibrate overnight.
04JAN18
I need to concentrate down the AKTA fractions where I detected the IRM12 protein (F11,12,13 and 14) to 2.5ml. The big AKTA size separation column can work with up to 5ml but it would be better to concentrate them to 2.5ml. This took the whole day spinning down at 4oC and topping up with more liquid. I kept the flowthrough.
We run the big AKTA with Annemette. I first filtered my protein extract using a filter and then I sucked it within a syringe and placed it onto the AKTA. I run a method. This will take about 6 hours.
05JAN
After running the column exclusion, I tool the samples where I believe my sample was (based on the pick on the screen of AKTA) and I run them on a gel (gel on page ). Unfortunately I couldnt see my protein on the gel. There might be a problem with the His tag of the protein and it cant stick to the His column. I could model the protein using Swiss model (or pyMol) to see the 3d structure of the protein and whether the His tag is in good position for binding to external substances. Also another idea is to run a natural gel (non denatured protein and try to place it). Since IRM13 seems to have very good purification results in gel on page 32 there is an idea that the concentration after purification (spinning down with the cut off filters at 4oC) affects the protein and after concentration the protein get lost or precipitated. So I will work with IRM13 but this time I will do dialysis overnight with the AKTA purification samples.
08JAN
Trying to find IRM12 I run all the samples from size exclusion AKTA on western (gel on page ). IRM12 was not found anywhere. It might got stuck in the column.
09JAN
I sonicated IRM13 and took the CFE. I kept the insoluble and some of the CFE for the gel later. I run the AKTA purification. The sample needs to get filtered before the AKTA. Also it is very good for buffer A to have some very low Imidazole concentration like 20mM. I took samples from ALL the AKTA samples before dialysis. I have noticed from gel on page 32 in combination with previous AKTA data stored in AKTA folder 'Deppie' that IRM13 elutes when buffer B is at 45% of the mixed buffer volume. So I took these samples (F20, F21, F22 and F23) and dialysed them overnight in buffer A.
10JAN
I run a gel and a western for all the AKTA samples (F1-F30), the CFE and insoluble fraction I have kept and the dialysed fractions F20D, F21D, F22D and F23D (gels on pages ).
***DISCREPANCY FOR WESTERN***
I didnt have PBS tablets and I used a PBS liquid that Harveen has on the self on her bench. It is 10x concentrate so I made it 1x (I put 60ml in 600ml of PBS buffer).
The gels were very good and the protein was found cleanest on F27, F28,, F29 and F30. So I decided to dialysed them overnight and work with them. Tomorrow I will calculate the concentration which i will get into some assays for pH and temperature. Since something always happens when the concentration is calculated, I will continue with the assays tomorrow and I calculate the concentration with BCA but without considering the BCA calculation essential for my assays. I will store some of the protein maybe in glycerol.
After coming back from festive break, I tool my samples from the freezer and run 2 gels containing all of the AKTA IRM12 fractions (see gels on pages 41 and 42). The IRM12 protein is detected in F11,12,13 and 14 while there is also unspecific binding of other proteins. So I will be doing another AKTA (one that I dont have access to and I will do it with Anemmette). Using this AKTA I will do a size separation using a column.
I made a 2lt of 50mM MES buffer A (recipe on page 116 DR 0817-073), 2lt of water and 1lt of Ethanol 20% and I degased them.
I wash the AKTA with water, and leave it with buffer A to calibrate overnight.
04JAN18
I need to concentrate down the AKTA fractions where I detected the IRM12 protein (F11,12,13 and 14) to 2.5ml. The big AKTA size separation column can work with up to 5ml but it would be better to concentrate them to 2.5ml. This took the whole day spinning down at 4oC and topping up with more liquid. I kept the flowthrough.
We run the big AKTA with Annemette. I first filtered my protein extract using a filter and then I sucked it within a syringe and placed it onto the AKTA. I run a method. This will take about 6 hours.
05JAN
After running the column exclusion, I tool the samples where I believe my sample was (based on the pick on the screen of AKTA) and I run them on a gel (gel on page ). Unfortunately I couldnt see my protein on the gel. There might be a problem with the His tag of the protein and it cant stick to the His column. I could model the protein using Swiss model (or pyMol) to see the 3d structure of the protein and whether the His tag is in good position for binding to external substances. Also another idea is to run a natural gel (non denatured protein and try to place it). Since IRM13 seems to have very good purification results in gel on page 32 there is an idea that the concentration after purification (spinning down with the cut off filters at 4oC) affects the protein and after concentration the protein get lost or precipitated. So I will work with IRM13 but this time I will do dialysis overnight with the AKTA purification samples.
08JAN
Trying to find IRM12 I run all the samples from size exclusion AKTA on western (gel on page ). IRM12 was not found anywhere. It might got stuck in the column.
09JAN
I sonicated IRM13 and took the CFE. I kept the insoluble and some of the CFE for the gel later. I run the AKTA purification. The sample needs to get filtered before the AKTA. Also it is very good for buffer A to have some very low Imidazole concentration like 20mM. I took samples from ALL the AKTA samples before dialysis. I have noticed from gel on page 32 in combination with previous AKTA data stored in AKTA folder 'Deppie' that IRM13 elutes when buffer B is at 45% of the mixed buffer volume. So I took these samples (F20, F21, F22 and F23) and dialysed them overnight in buffer A.
10JAN
I run a gel and a western for all the AKTA samples (F1-F30), the CFE and insoluble fraction I have kept and the dialysed fractions F20D, F21D, F22D and F23D (gels on pages ).
***DISCREPANCY FOR WESTERN***
I didnt have PBS tablets and I used a PBS liquid that Harveen has on the self on her bench. It is 10x concentrate so I made it 1x (I put 60ml in 600ml of PBS buffer).
The gels were very good and the protein was found cleanest on F27, F28,, F29 and F30. So I decided to dialysed them overnight and work with them. Tomorrow I will calculate the concentration which i will get into some assays for pH and temperature. Since something always happens when the concentration is calculated, I will continue with the assays tomorrow and I calculate the concentration with BCA but without considering the BCA calculation essential for my assays. I will store some of the protein maybe in glycerol.
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