Histidine tag detection by antibodies
Either to confirm the expression of your desired recombinant protein using Western Blot or to easily purify it, Histidine tag 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.
Construction of recombinant protein with His Tag for easy purification and detection
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, patent expired in 2003). This is the kind of work many labs routinely performed, including SYnAbs with sister company RD-Biotech.
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 of 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.
|
SYnAbs #013 |
SYnAbs #007 |
SYnAbs #010 |
SYnAbs #030 |
#1 Top Reference |
DMEM | |
| Influenza membrane protein | 3,300 | 3,100 | 2,500 | 2,600 | 2,150 | 0,110 |
| Metallo-B-lactamase #1 | 3,200 | 3,100 | 2,300 | 2,300 | 1,890 | 0,100 |
| Nucleocapsid protein | 0,180 | 0,370 | 2,900 | 2,900 | 1,700 | 0,120 |
| Surface glycoprotein #1 | 3,300 | 3,400 | 0,150 | 0,140 | 0,600 | 0,110 |
| Surface glycoprotein #2 | 3,600 | 3,300 | 3,300 | 3,100 | 1,900 | 0,100 |
| Surface glycoprotein #3 | 3,500 | 3,300 | 3,300 | 3,100 | 1,900 | 0,100 |
| Toxin | 0,180 | 0,330 | 1,500 | 1,500 | 1,900 | 0,100 |
| VHH #1 | 3,300 | 2,900 | 3,500 | 3,300 | 2,500 | 0,120 |
| VHH #2 | 3,100 | 2,700 | 1,700 | 1,500 | 1,600 | 0,120 |
| Metallo-B-lactamase #2 | 2,700 | 2,300 | 0,600 | 0,400 | 0,160 | 0,120 |
|
Metallo-B-lactamase #2 w/o His Tag |
0,300 | 0,300 | 0,180 | 0,180 | 0,180 | 0,120 |
| Binding protein #1 | 3,300 | 3,300 | 3,100 | 3,000 | 2,100 | 0,120 |
| Binding protein #2 | 2,700 | 2,800 | 2,600 | 2,700 | 1,800 | 0,120 |
| Mannose receptor | 0,300 | 0,600 | 2,700 | 2,400 | 1,700 | 0,120 |
| Surface glycoprotein #4 | 2,900 | 2,900 | 2,800 | 2,700 | 0,640 | 0,120 |
| Metallo-B-lactamase #3 | 3,100 | 3,000 | 2,600 | 2,400 | 3,300 | 0,120 |
| Metallo-B-lactamase #4 | 3,100 | 2,900 | 2,400 | 1,800 | 0,200 | 0,120 |
| Metallo-B-lactamase #5 | 3,400 | 3,300 | 2,300 | 2,400 | 0,200 | 0,120 |
| Control | 0,120 | 0,130 | 0,130 | 0,150 | 0,150 | 0,120 |
| SUCCESS RATE | 14/17 | 14/17 | 15/17 | 15/17 | 11/17 | |
|
SYnAbs #013 |
SYnAbs #007 |
SYnAbs #010 |
SYnAbs #030 |
#1 Top Reference |
Even though our monoclonal antibodies showed better results compared to the #1 competing reference on the market, we still wanted to detect any recombinant protein. So we considered the results a semi-failure (or semi-success, depending on your philosophy of life).
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?
You'll find below the ELISA outcomes we finally obtained:
|
SYnAbs cocktail #007/#010 |
#1 Top Reference |
DMEM | |
| Influenza membrane protein | 3,300 | 2,150 | 0,110 |
| Metallo-B-lactamase #1 | 3,200 | 1,890 | 0,100 |
| Nucleocapsid protein | 2,900 | 1,700 | 0,120 |
| Surface glycoprotein #1 | 3,300 | 0,600 | 0,110 |
| Surface glycoprotein #2 | 3,600 | 1,900 | 0,100 |
| Surface glycoprotein #3 | 3,500 | 1,900 | 0,100 |
| Toxin | 1,500 | 1,900 | 0,100 |
| VHH #1 | 3,500 | 2,500 | 0,120 |
| VHH #2 | 3,100 | 1,600 | 0,120 |
| Metallo-B-lactamase #2 | 2,700 | 0,160 | 0,120 |
|
Metallo-B-lactamase #2 w/o His Tag |
0,180 | 0,180 | 0,120 |
| Binding protein #1 | 3,300 | 2,100 | 0,120 |
| Binding protein #2 | 2,700 | 1,800 | 0,120 |
| Mannose receptor | 2,700 | 1,700 | 0,120 |
| Surface glycoprotein #4 | 2,900 | 0,640 | 0,120 |
| Metallo-B-lactamase #3 | 3,100 | 3,300 | 0,120 |
| Metallo-B-lactamase #4 | 3,100 | 0,200 | 0,120 |
| Metallo-B-lactamase #5 | 3,400 | 0,200 | 0,120 |
| Control | 0,120 | 0,150 | 0,120 |
| SUCCESS RATE | 17/17 | 13/17 | |
|
SYnAbs cocktail #007/#010 |
#1 Top Reference |
