The nanobody I have used was selected as a binder of the intact GFP 3D-structure which will probably be mostly denatured in a Western Blot, so you won't get any nice signal if any at all. See Kubala et al., Protein Sci. 2010 Dec; 19(12): 2389–2401.

Why not use a normal antibody?

Hi Huseyin,

Thanks for the reply. I have heard these nanobodies are successfully used in westerns. I wanted to give it a try since westerns with regular GFP antibodies (at least the ones I have seen) do not usually give clean westerns specially for band quantification. Reference is helpful.

Hi folks, could anyone share some good references or reasons why GFP is denatured and not easy to expressed in e.coil? I have linked a GFP to a nanobody and when I run the Protein on Western blot I got it fragmented and was not able to purify it. Would it be possible to get my expected protein if I try different expression protocols or this is not going to work.. I am interested to know about other experiences though.

Also, would any one suggest some researchers in the field of protein activity and structure who may be interested to help out in a research for determine an epitope on a specific protein?

Another question please. Did anyone try to successfully conjugate a nonobody to HRP?

Nahid Nasir you should post independent questions as a new post, not in comments to another question.

But - you should use superGFP or super-folder GFP. These fold well in E. coli. You can find sequence on Addgene I believe, if not, then on PDB.