| Background |
Originating from the influenza virus, the hemagglutinin (HA)-epitope (YPYDVPDYA) has become a popular fusion tag for mammalian recombinant protein expression over the last years.
A high-quality monoclonal Anti-HA antibody is offered by Miltenyi Biotec either coupled to µMACS™ MicroBeads for specific isolation of HA-tagged proteins or conjugated to biotin, FITC, PE, or horseradish peroxidase (HRP) for analysis of the recombinant, HA-tagged protein. |
| Isolation and detection of HA-tagged proteins |
Isolation of HA-tagged proteins
The µMACS™ and MultiMACS™ HA Isolation Kits take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure HA-tagged proteins (fig. 1). The advantages of MACS Technology for protein isolation are:
Specificity – high-quality monoclonal antibodies coupled to MACS MicroBeads assure specific isolation of HA-tagged proteins with low background.
Sensitivity – fast reaction kinetics of tiny (50 nm), non-sedimenting MACS MicroBeads allow sensitive isolation of even when working with rare proteins.
Speed – less than 2 hours to high-purity HA-tagged protein!
The µMACS HA Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator.
The procedure can easily be upscaled with the MultiMACS HA Isolation Kits to a semi- or fully-automated, high-throughput processing of up to 96 samples in parallel by utilizing the MultiMACS 96 Separator.
Detection of HA-tagged proteins
The high-quality monoclonal Anti-HA antibody is available with various conjugates.
The fluorochrome-conjugated (FITC or PE) Anti-HA antibodies enable an immunofluorescence analysis e.g. by fluorescence microscopy (fig. 2), whereas the Anti-HA-HRP antibody was generated for specific and sensitive detection of HA-tagged proteins by Western blot or ELISA analysis. Directly coupled to horseradish peroxidase (HRP), the Anti-HA-HRP antibody simplifies protocols as incubation with secondary antibodies is not necessary.
Varied detection methods are applicable using the biotinylated Anti-HA antibody in combination with appropriate streptavidin conjugates.
Antibody specifications
Target sequence: YPYDVPDYA
Tag origin: influenca virus hemagglutinin
Isotype: mouse IgG1 |
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| Figure 1 |
| Sensitive isolation of recombinant fusion protein with μMACS™ Anti-HA MicroBeads. 107 mouse pre-B cells (1881) were transfected with a vector encoding HA-BDCA-2 and proteins isolation was performed using cell populations with either 1% (lane 1, 3) or 10% (2, 4) positively transfected cells. Whole-cell lysates (1, 2) or 20% of the eluate from a protein isolation with Anti-HA MicroBeads (3, 4) were separated by SDS-PAGE, blotted on a membrane and detected by using Anti-HA-HRP. |
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| Figure 2 |
| Immunofluorescence analysis of HA-BDCA-2 expressing RBL-1 cells. RBL-1 (rat basophilic leukemia) cells were transiently transfected with a vector encoding HA-BDCA-2 and analysed by confocal fluorescence microscopy after 24 hours of expression. Cells were first labeled with CD303(BDCA-2)-Biotin, fixed, permeabilized, and then stained with Anti-HA-FITC and Streptavidin-Alexa633. A Fluorescent confocal image of transfected RBL-1 cells. B Overlay of transmission image and fluorescent confocal image of transfected RBL-1 cells. |
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