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His-tag protein lost it's tag


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#1 Hsiao

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Posted 28 May 2010 - 05:41 PM

Hi,

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

R.Hsiao

#2 pDNA

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Posted 28 May 2010 - 10:40 PM

are you trying to detect it in its native or denatured form?

could be that the his-tag is somehow hidden due to the conformation of the protein.

Regards,
p

#3 HomeBrew

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Posted 29 May 2010 - 04:34 AM

If you've checked the sequence and the fusion + insert sequence is correct, then cloning it into another vector probably won't help. Is this a C-terminal or N-terminal fusion? If it's N-terminal, could it be that your protein has some kind of N-terminal signal sequence that's cleaved after translation? Maybe you could try a C-terminal fusion instead.

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...




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