Zdravím :)

I don't think the problem is due to few introduced amino acids. I've heard about problems with detecting His-tag with commercial antibodies, so that could be one reason. The other could be that the His-tag is not accessible for antibody. I guess you have purified your protein on Ni-NTA, right?

Ahoj,

inaccessibility is the main reason, therefore even IMAC purification is not working for our molecules. Seem to be a rule of thumb, whether it is accessible or not, given the fact, some papers reported purification/detection of His-tagged scFv. My question was more of an curiosity, if other labs experience similar issue.

are your recombinant antibodies scFvs? try to have them as periplasmic proteins. we usually use sv5 tag but when we had to use his tag (pET -based vectors) we did not face real problems

I would agree that accessibility is an issue. For Western blots we always use a NuPAGE Bis Tris gel.

Tomáš Hluska is right. Inaccessible is the main reason. I am myself working on transmembrane protein and I find it difficult to find literature of western from lysate until protein is purified. I have faced similar problems too but upon standardisation and using good vector, my problem is solved to some bit.

@Claudio

Yes, they are expressed as periplasmic protein (pET22). Changing the His-tag to myc-tag resolved the detection issue, but it is more inconvenient to handle purification without His-tag, you know what I mean. What is the format of your scFv regarding chain order and flanking restriction sites? Do you fuse His-tag directly to c-terminal of an antibody ?

hi

There is a possibility that the His tag is hidden in the folds of your protein. U can treat the protein with urea or ant other mild detergents which denature the antibody and expose the hidden grooves and thus the his tag too.... All the best.