We have designed a surface associated protein (Arg-specific cysteine proteases) in such a way that it will be secreted in culture broth. For easy purification we have inserted His-Tag and His-Tag preceded by Thrombin separately at the C-terminal region of that protein. We have inserted encoding gene using SOE primer and confirmed by sequencing. After ligated with plasmid, transformed in E. coli, then further confirmed that transformed cells are cloning plasmid with Hi-Tag and His-Tag preceded by Thrombin. Finally after digestion of plasmid with BgLI, we transformed plasmid in P. gingivalis and confirmed transformed cell by PCR and both transformed cell are secreting protease. We further confirmed protein by SD-PAGE and observing proteolytic activities. To know if both proteins are carrying His-Tag and Thrombin-His-Tag, we mixed vesicles free supernatants of culture broth with Ni resin for 1 hour at 40C with swirl. Then centrifuged and discarded supernatants, after that washed twice with binding buffer (50 mM Tris, 300 mM NaCl and 20 mM imidazole, pH 7.5). Finally added 500 mM imidazole and centrifuged, took supernatant and run SD-PAGE. We did not get any protein on the gel. I would like to know is there any simple method by which I can confirm that my protein still bearing His-Tag?
you can use anti-His monoclonal or even polyclonal antibody. western blot or dot blot help you to distinguish the His-tagged protein. diaminobenzidine+H2O2 determines proteins with His-tag.
I agree with the suggestions given by Fatemeh. In addition, you need to consider the conditions of your binding and/elution buffers, particularly pH and salt concentration affects binding.
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