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Anti Histag antibody vs. Referee

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#1 Ivanov_br



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Posted 10 May 2011 - 10:08 AM

This is the kind of topic we always get a bit confused where to post it.

Well, I cloned, expressed and purified a mamalian protein (his-taggeg)in E. coli. There is no comercial Antibody avaiable for it, so to test its ability to bind to cells, I used western blotting and imunofluorescence microscopy with Anti Histag as primary antibody. Assays were based on incubation of cells with the recombinant protein, and results showed that the protein binds to cell, and the microscopic analysis showed an unique binding pattern. My controls were NEGATIVE (cells incubated without protein) and NEGATIVE HIS (cells incubated with another histagged protein).

However the referre said my results were all artfacts because I did not use an Antibody against the protein itself.

Do you think that my results with anti histtag are really that fragile?

In the picture, a western blotting showing my results with incubation with different concentrations of purifyed recombinant protein. I did the incubation as described elsewhere and after washing I ran the cell pellet in 12% SDS-PAGE for WB. Numbers indicate concentration of recombinant proteins in ng, PBS means negative control (without protein), and positive control is the recombinant protein in the lane.

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  • Figure4 copy.jpg

Edited by Ivanov_br, 10 May 2011 - 10:13 AM.

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