PCMP-H8 Antibody
Description
What Is the HIS.H8 Antibody?
The HIS.H8 antibody is a mouse monoclonal antibody that binds specifically to the 6x-His epitope (HHHHHH), a synthetic tag commonly fused to recombinant proteins for purification and detection . It is used extensively in molecular biology and biochemistry for:
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Western blotting to validate His-tagged protein expression.
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Immunoprecipitation to isolate tagged proteins.
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Immunocytochemistry/immunofluorescence to localize tagged proteins in cells .
Key Features:
| Property | Detail |
|---|---|
| Target | 6x polyhistidine (6x-His) tag (N-terminal, C-terminal, or internal) |
| Specificity | Recognizes HHHHHH motif regardless of protein sequence or fusion position |
| Applications | Western blot, immunoprecipitation, ICC/IF, ELISA |
| Purity | >90% (SDS-PAGE), <10% aggregation (HPLC) |
| Conjugation | Unconjugated (primary antibody) |
Mechanism of Action
The HIS.H8 antibody binds to the 6x-His tag via non-covalent interactions, enabling its use in downstream applications such as:
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Purification: Facilitates immobilized metal affinity chromatography (IMAC) using nickel or cobalt columns .
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Detection: Serves as a primary antibody in western blotting, with secondary antibodies (e.g., anti-mouse IgG-HRP) amplifying signal .
Example Workflow:
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Protein Expression: Recombinant protein with 6x-His tag expressed in E. coli or mammalian cells.
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Purification: Nickel-NTA column binds His-tagged protein.
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Detection: Western blot using HIS.H8 antibody (1:500–1:5000 dilution) confirms successful expression .
Research Applications and Findings
The HIS.H8 antibody has been cited in over 350 publications, primarily for validating His-tagged proteins in diverse systems:
3.1. Viral Protein Studies
In COVID-19 research, HIS.H8 was used as a loading control for western blots analyzing SARS-CoV-2 spike glycoprotein (S1) and nucleocapsid proteins. For example:
| Protein | Application | Result |
|---|---|---|
| SARS-CoV-2 Spike S1 | Western blot validation | Detected 135 kDa band in transfected cells |
| NSP2 (nucleocapsid) | Coomassie-stained gel confirmation | Confirmed His-tagged protein purity |
3.2. Cellular Localization
In immunofluorescence studies, HIS.H8 antibody (1:1000 dilution) successfully localized His-tagged proteins in HEK293 cells, with Alexa Fluor 488-conjugated secondary antibodies enabling visualization .
Comparative Analysis of HIS.H8 Antibodies
| Vendor | Product Code | Dilution (WB) | Applications Tested | Citations |
|---|---|---|---|---|
| Thermo Fisher | 14-6657-82 | ≤1 µg/mL | Western blot, IP | N/A |
| Abcam | ab18184 | 1:500–1:5000 | WB, ICC/IF, IP | >350 |
| Antibodies.com | A85277 | 1:500 | WB (SF9 cell lysates) | N/A |
Data aggregated from product sheets .
Considerations and Limitations
Product Specs
Components: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
