Either to confirm the expression of your desired recombinant protein using Western Blot or to easily purify it, tag Histidine has become a relatively used technique amongst the biotech community.
A poly-histidine tag is an amino acid motif consisting of several histidine residues, inserted either at the N- or C-terminus of the protein. It is frequently used for:
- purification by IMAC (Immobilized Metal ion Affinity Chromatography) of proteins produced in E.Coli expression system,
- protein-protein interactions,
- fluorescent dyes to follow the circulation of specific proteins.
Within the expression vector, and just upstream the transgene, it’s possible to insert six histidine codons encoding the peptide tag (invented and patented by Roche in 2003). This is the kind of work many labs routinely performed including SYnAbs with sister company RD-Biotech for a specific project.
Nevertheless, testing available references on the market, we were deeply disappointed since so many primary monoclonal antibodies couldn’t detect specifically and effectively His-tag residues. Very often, Western-Blot couldn’t give any signal at all or during the purification step the protein of interest would not bind to the resin.
In France, we have a proverb that says “you are never better served than by yourself”. So we decided to develop a new rat monoclonal antibody reference targeting N-terminal and C-terminal parts of histidine and tested it against 17 different recombinant proteins, covering the entire spectrum of proteins such as enzymes, membrane protein, viral capsid, VHH antibodies, binding proteins.
Generating 30 different clones, these are the outcomes we obtained with the best of them.
To be frank, our antibodies were better than the #1 competitor reference on the market, but we still wanted to detect any recombinant protein. So it was a semi-failure (or semi-success, it depends on your life philosophy ;-). After so many fusions, our conclusion was it’s not possible to generate a monoclonal antibody targeting His-tag on any protein.
And then, we figured that a synergetic cocktail of our best monoclonal antibodies should potentially do the job. If 1 monoclonal couldn’t do the job, maybe 2 could?
And these are the ELISA outcomes we finally obtained: