Hi there,
I am trying to purify a recombinant His-tag protein from insect cell (baculovirus expression system) I used buffer containing %10 glycerol, 50mM SODIUM PHOSPHATE, 300mM NaCl , 20mM imidazole PH:8 , protein PI is 9. and went through sonication (10 sec on/1min off for 10cycles ). Sample was centrifuged at 16000rpm and finally filtered with 0.45 Micron. I used his trap column for purification (washing buffer 50mM SODIUM PHOSPHATE, 300mM NaCl , 20mM imidazole PH:8 elution buffer (50mM SODIUM PHOSPHATE, 300mM NaCl , 500mM imidazole PH:8 . Protein was dialyzed with amicon ultra 15 (100 kda). The size of protein is 234 kda. However, there is a bunch of unspecific bands along with the protein of interest even after dialysis.
Can anyone who has successfully purify recombinant protein from insect cell give me suggestions as to the best way to get rid of unspecific bands?
There are several things you should check.
1. Was the protein expressed? Compare infected versus uninfected cell lysates by Western blot. This will also help you determine whether the anti-His antibody is detecting background bands from the cells.
2. If expressed, was the protein intact or proteolyzed or truncated? Check by Western blot in the same experiment as above.
3. Were the cells adequately broken? Check under the microscope.
4. Was the protein in the supernatant or the pellet after centrifuging the lysed cells?
5. Did the protein stick to the affinity resin, or just flow through.
There are several things you should check.
1. Was the protein expressed? Compare infected versus uninfected cell lysates by Western blot. This will also help you determine whether the anti-His antibody is detecting background bands from the cells.
2. If expressed, was the protein intact or proteolyzed or truncated? Check by Western blot in the same experiment as above.
3. Were the cells adequately broken? Check under the microscope.
4. Was the protein in the supernatant or the pellet after centrifuging the lysed cells?
5. Did the protein stick to the affinity resin, or just flow through.
I think your protein is poorly expressed . Please check the expression level.
From the gel image, it looks as if your protein isn't adequately expressed. After sonication, run SDS-PAGE to check whether the protein is in the pellet or supernatant.
I agree with dear colleagues Adam B Shapiro Tushar Chakraborty
There are many questions about photography. And what do you get at the exit. If it's a mixture of protein and gel this is one thing. If the protein is not cleared from the cell contents, this is another solution.
Regards, Sergey
Hi,
You can always add a size exclusion step och a IEX step after the first Ni-charged resin. How pure do you need your protein? You seldom get very high purity with just one IMAC column.
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