I want to purify a secreted, HIS-tagged 24 kD protein from a culture supernatant of Hek 293T cells. If I wanted to remove the BSA from the medium, what level of saturated ammonium sulfate should I used for BSA precipitation?
If the protein is His Tagged and you are using a Ni column purification to pull it out , then I am not clear why you need to ppt BSA ? Not knowing anything about your protein, it is not possible to say if adding sat ammonium sulphate will not also bring down your protein of interest. If the issue is BSA binding to your column, then try binding in more stringent conditions
Ian Teo
Hi ,thanks for your answer. I wanted to purify my protein from HEK293 condition media using the His select affinity gel from sigma. but I saw the BSA band on the SDS PAGE . and I didn't see my protein band. so I decided to remove BSA from media.
Hi,
What is the MW of your His-tagged protein? If its too close from BSA you will precipitate both of them. For BSA removal you can use some commercial resins that selectively bind BSA (something like Blue-sepharose column). Otherwise you might have to optimize the purification steps with your Ni column (height, imidazole concentration, ...)
Thomas
hi and thanks . I tried to optimize the purification but unfortunately I havnt a good protocol for purification of his tagged protein from condition media :(
If you are seeing some of the albumin in the eluate with your tagged protein, try passing the eluate through a cibacron blue resin. Albumin binds with high affinity to cibacron blue. Your His tagged protein should be in the flowthrough unless it also binds to the cibacron blue, which would be pretty unlucky.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression