His-tag protein lost it's tag
Posted 28 May 2010 - 05:41 PM
I have cloned a gene cDNA to pQE30 vector and checked the coding sequence which were all in frame.
When I expressed the protein in JM109, I couldn't detect it with anti-6x His Ab in any fractions of wash
buffer or elution product after I purified the protein from Ni-NTA column.
But when I use it's specific Ab, I got a strong signal in wash buffer but not elution product.
Could this be due to the lost of His-tag during protein induction ?
Should I cloned this protein into another vector ?
Any advice greatly appreciated
Posted 28 May 2010 - 10:40 PM
could be that the his-tag is somehow hidden due to the conformation of the protein.
Posted 29 May 2010 - 04:34 AM
I think you're saying that your backbone-speciifc Ab detected the protein in your wash buffer, but the anti-6x His Ab did not, correct? Did you run samples of the wash buffer on SDS-PAGE and screen by western blot, or just check them by dot blot? If it's a protein conformation problem, perhaps the screen would be negative by dot blot, but be detectable when the protein is in denatured form after SDS-PAGE.
Do you know that your anti-6x His Ab works? We've had some problems with such antibodies -- in some experiments one vendor's anti-6x His MAb doesn't work while another vendor's does...