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His-tagged scFv

scFv antibody histidine tag detection

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#1 matoxxx

matoxxx

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Posted 15 May 2012 - 12:04 AM

Hello fellow netters,

I have a question for people in field of recombinant antibodies. For some reason, we are unable to get active c-terminal 6x His-tag, when recombinant antibody is expressed in E.coli, confirmed either by ELISA or WB. Protein is expressed in correct length, confirmed by MALDI-TOF. My feeling tells me , this might be due to the c-terminal sequence (Ala-Ala-Ala-Leu-Glu), preceding His-tag, introduced by cloning of an antibody.
Nevertheless there are other publications on net, without problems with active His-tag, directly in frame with c-terminus of antibody. Might be the result of common process, not to publish, what is not working.
If anybody experienced similar issues, please share them with me.

M.





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