Hi there.
I have to use the pET28a-SUMO vector to get my target protein.
However, I can‘t separate it from SUMO tag.
My protein was about 15 Kda big and has a about 5 value of pI.
Due to the process next is about X-ray diffraction.
How I can i get my protein without tag or without 80% tag？
After tag on/off-columns cleavage, my protein will stick on the Ni column if I want to remove the tag only if I introduce buffer with imidazole. But buffer with imidazole will bring the SUMO tag down even you use 10mM imidazole.
How can i get my protein without SUMO tag ??????
I have tried ion exchange chromatography and It was not work.
6His tag only? Inclusion body.
MBP tag?TEV is trash enzyme and same problem when i remove the tag.
GST tag?A little of protein is soluble.
The statement I don't understand is that your protein will only stick to the Ni column when imidazole is present. That's odd.
If the tag is cleaved and the protein sticks to the column in the presence of imidazole, for whatever reason, the tag should not stick to the column, so you should be able to separate them by binding the protein to the column in the presence of imidazole. You then have to elute the tag-free protein from the column, probably by raising the imidazole concentration, or lowering it (?).
Can you clarify the phenomena you are seeing a bit more.
Hello
Use Size Exclusion Chromatography (SEC), which separates proteins based on molecular weight.
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