Histidine tag detection by antibodies
Monoclonal antibodies anti-His tag have become an essential tool in biotechnology for detecting recombinant proteins. Whether confirming the expression of your desired protein using Western Blot or simplifying its purification, the histidine tag technique is now widely adopted within the scientific community.
A poly-histidine tag is an amino acid sequence made up of several histidine residues, inserted at either the N- or C-terminus of a protein. This tag is commonly used for the following purposes:
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Protein purification using Immobilized Metal Ion Affinity Chromatography (IMAC), particularly for proteins produced in the E. coli expression system.
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Protein-protein interactions.
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Utilization of fluorescent dyes to track specific proteins in circulation.
Constructing Recombinant Proteins with His Tag for Easy Purification and Detection
Within the expression vector, it’s possible to insert six histidine codons encoding the peptide tag just upstream of the transgene. This system, invented and patented by Roche, has been in the public domain since the patent expired in 2003.
However, after testing several commercial references, we were deeply disappointed by how many monoclonal antibodies anti-His tag failed to detect His-tag residues specifically and effectively. In many cases, Western Blot failed to produce any signal, or during the protein purification step, the protein of interest would not bind to the resin.
As the French proverb goes, “You’re never better served than by yourself.” This is why we decided to develop a new rat monoclonal antibody targeting both the N-terminal and C-terminal regions of the histidine tag. We tested it on 17 different recombinant proteins, covering a broad range of proteins, including enzymes, membrane proteins, viral capsids, VHH antibodies, and binding proteins.
Impressive Results with Anti-His Tag Monoclonal Antibodies for Precise Detection
By generating 30 different clones, we achieved remarkable results with the best of them. These monoclonal antibodies anti-His tag demonstrated not only superior detection capabilities for recombinant proteins carrying the His tag but also high efficiency during the purification step.
Our anti-His tag monoclonal antibodies are now available for research use only (RUO) and are a valuable asset for any study involving recombinant proteins.
|
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 |
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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 |
While our monoclonal antibodies outperformed the top competitor on the market, we were still keen on detecting any recombinant protein. So, we took the results as a bit of a 'semi-failure' (or 'semi-success,' depending on your outlook on life). Then, we had an idea: what if a combination of our best monoclonal antibodies could do the trick? If one couldn’t cut it, maybe two would?
Below, you'll find the ELISA results we ultimately achieved:
|
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 |
