His-tag is a series of six to nine histidine residues fused to either the carboxyl or amino terminus of a recombinant protein. The small size of the His-tag, compared with other common epitope tags, makes it less likely to obstruct the target protein’s structure or function and more suitable to use under denaturing conditions. The string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper. The binding to metal ions under specific buffer conditions allows for the simple purification and detection of His-tagged proteins. His-tag antibodies provide a dependable method for the detection and purification of tagged target proteins without a protein-specific antibody or probe. Our His-tag antibodies also come with the option of being HRP, biotin, or fluorophore conjugated to meet all your research needs. Invitrogen His-tag antibodies reliably detect recombinant His-tagged proteins and are validated for use in various applications.

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Applications

Invitrogen His-tag antibodies are validated for use in applications such as ELISA, western blot, flow cytometry, immunofluorescence, immunohistochemistry, immunocytochemistry, and immunoprecipitation.

Immunofluorescence analysis of Histidine tag (6x-His) was done on HEK-293 cells. Left: Untransfected control; Right: anti-His (in red) on His-tagged fusion proteins in HEK293 cells. The cells were labeled with 6x-His Tag Monoclonal Antibody (HIS.H8), HRP (Cat. No. MA1-21315). Both counterstained with DAPI (in blue). Rabbit anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Flow cytometry analysis of Histidine tag (6x-His) was done on HEK-293 cells transiently overexpressing His-IGFBP6. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with 6x-His Tag Monoclonal Antibody (4A12E4) (Cat. No. 372900, red histogram) or with mouse isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor 488.

Flow cytometry detection of Histidine tag using 6x-his mouse monoclonal antibody
Western blot analysis of 6x-His Tag using 6x-His Epitope Tag monoclonal antibody

Western blot analysis of 6x-His Epitope Tag was performed by loading various amounts of E. coli lysate, containing a multi-epitope tagged protein, per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an 6x-His Tag Monoclonal Antibody (HIS.H8), HRP (Cat. No. MA1-21315-HRP) at a dilution of 1:1000 overnight at 4oC on a rocking platform and washed in TBS-0.1% Tween™ 20. Chemiluminescent detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Cat. No. 34580).

Resources

For Research Use Only. Not for use in diagnostic procedures.