I have successfully expressed and purified target protein using pTXB1 vector from NEB. We confirmed the desired molecular weight by SDS PAGE and identity of the protein by mass spectrometry. To elute the target protein from the chitin resin, we incubated the resin with a thiol cleavage buffer (50 mM Tris pH 8.0, 150 mM NaCl, 100 mM MESNA) for about 40 hours at room temperature and 37 C. As expected the target protein should elute from the resin following Thiol-induced cleavage with the Intein-CBD tag still bound to the chitin resin. However, we see that the Intein-CBD tag coming off the chitin resin following the thiol-induced cleavage with a little or no free target protein. What could be the reason for this problem? Has anyone encountered this issue before. Your assistance in this regard will be highly appreciated.
Hi @Prasad Sulkshane
I have the same problem these days.
Did you solve this problem? Could do you please give me some advice?
Thank you very much!
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