Hi,
My recombinant protein of size ~ 20kDa has a 6X His tag at its C-Terminal end.
This recombinant proteins` purpose is to serve as a diagnostic antigen looking for antibody in the infected individual
Can western blotting/ELISA be done for my recombinant protein? Does the His interferes in antibody interaction?.
Thank you
His-tag never interfere with Western Blot, actually you can use Anti-His to detect proteins with His-tag when the expression levels are too low to be detected with Comassie staining.
Thank you Gopal and Sanfelice.
@Sanfelice: Yes true i can use Anti-His, However, my objective is to use this recombinant protein as a diagnostic antigen. This HIS tagged recombinant protein could probably be coated onto ELISA plates or onto the membrane strips for western blotting. So this HIS should not interfere with the diagnosis.
@Gopal: Just curious to know : Is there any curoff kDa size for HIS tagged recombinant protein that below some kDa the 6XHIS will interfere. [since you replied 20kDa shall not interfere]
If the HIS tag is not important for solubility and stability of your target protein, you can remove the tag by enzyme-mediated cleavage followed by purification by size exclusion chromatography. You can separate your 20kDa protein from HIS tag by using Sephadex s75 column
The 6x His tag should not be a problem for Western/ELISA. I have used it for proteins which were 35 kDa and 82kDa and it worked with both (in case you think there is a size constraint).
May be you can delete His-tag by enzyme-mediated cleavage followed by purification by size exclusion chromatography。
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