I have a protein expressed in mammilian cells and it has a 6x-HIS tag on it and it is supposed to be secreted but I can't really find it in the supernatant by Western. I can however see it in the cell lysate by Western. I was thinking of using the MagneHis Protein Purification System from Promega but I haven't found any journals that show people have used this system. I was also thinking of using the His Mag Sepharose Excel, but I don't know.
hi
before ordering any kit you should check medium in WB for right expression of your interest protein by specific antibody or anti-His -HRP antibody . other methods, IP with Agarose Ni-NTA bead for culture medium.
you can uesd Ni Sepharose 6 Fast Flow .
good luck
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-us/17531801
We use these beads and they work well for this application (with the right media and good expression we can purify enough for coomassie staining) but the fact that you don't see it by Western except in the cell lysate suggest that it is not being efficiently secreted.
Have you tried a time course leaving the cells up to a week (normally we harvest our secreted proteins at day 4 or 5), different proteins can take different times to be secreted? Also check that you have cloned either the whole protein including signal peptide and signal peptidase site or included a signal peptide/peptidase site from your vector.
Hi marsha how r u? simple way may be Ni-NTA sepharose. just mix the protein with the beads for 2-3h. load onto the column and follow the purification process. It should work.
Hi Raj! Its been a long time! I am doing well! How are you? Thanks for the info! :)
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