12-16FEB
12FEB
I degassed 2lt of Buffer A for IRM12 and 2 lt of water for washing the size exclusion column and equilibrate it over night
13FEB
I sonicated IRM12, got the CFE, and run the AKTA. This time I put 20mM of Imidazole in buffer A to minimise the unspecific binding I usually have (thats why the size exclusion separation is needed).
I could see any pick in the AKTA graph and I therefore run all the samples on the gel and left it for staining (see page for the gel).
***NOTE: For making the SDS samples
INSOLUBLE FRACTION: 10μl sample (as usual following the recipe)
CFE: 5 μl sample + H2O
AKTA FRACTIONS: 25μl sample
I load 10μl of the Insoluble and CFE while I load 20μl of the AKTA FRACTIONS
14FEB
Before using the size exclusion I need to be sure that my protein is the one I see on the gel (page )
So I run a western using the samples from yesterday. The IRM12 his tagged protein was found in the last AKTA FRACTIONS (surprisingly, see the western on page ) so I spun the fractions 24-27 for making 5ml for the size exclusion column. I put the samples to the size exclusion column overnight.
I did an activity check to the CFE (produced yesterday) following the instructions on page 55 (see image below)
15FEB18
I received the samples from the size exclusion and I run them on a gel for both blue staining and western. The samples which possibly contain the protein are the samples which present the UV pick on the graph. Unfortunately the pick inn conductivity coincides with the pick in the UV which means that Imidazole is the one that produces the the absorbance in UV and not my protein. The western and sds gel are both empty (see gels on pages ). Maybe there has been a problem with the sonication as the insoluble fraction seems to have a lot of protein compare to the CFE (see gel ).
I degassed 2lt of Buffer A for IRM12 and 2 lt of water for washing the size exclusion column and equilibrate it over night
13FEB
I sonicated IRM12, got the CFE, and run the AKTA. This time I put 20mM of Imidazole in buffer A to minimise the unspecific binding I usually have (thats why the size exclusion separation is needed).
I could see any pick in the AKTA graph and I therefore run all the samples on the gel and left it for staining (see page for the gel).
***NOTE: For making the SDS samples
INSOLUBLE FRACTION: 10μl sample (as usual following the recipe)
CFE: 5 μl sample + H2O
AKTA FRACTIONS: 25μl sample
I load 10μl of the Insoluble and CFE while I load 20μl of the AKTA FRACTIONS
14FEB
Before using the size exclusion I need to be sure that my protein is the one I see on the gel (page )
So I run a western using the samples from yesterday. The IRM12 his tagged protein was found in the last AKTA FRACTIONS (surprisingly, see the western on page ) so I spun the fractions 24-27 for making 5ml for the size exclusion column. I put the samples to the size exclusion column overnight.
I did an activity check to the CFE (produced yesterday) following the instructions on page 55 (see image below)
15FEB18
I received the samples from the size exclusion and I run them on a gel for both blue staining and western. The samples which possibly contain the protein are the samples which present the UV pick on the graph. Unfortunately the pick inn conductivity coincides with the pick in the UV which means that Imidazole is the one that produces the the absorbance in UV and not my protein. The western and sds gel are both empty (see gels on pages ). Maybe there has been a problem with the sonication as the insoluble fraction seems to have a lot of protein compare to the CFE (see gel ).
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