Isolation and analysis of HA-tagged proteins ( #130-091-122 #130-091-286 #130-092-256 #130-092-257 #130-092-258 #130-091-972 #130-094-255 #130-094-257 )

Isolation and analysis of HA-tagged proteins

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

Columns

For µMACS™ Isolation Kit: µ Column, upscale with M Column

For MultiMACS™ Isolation Kits: Multi-8 or Multi-96 Columns

Further information

Protein research brochure.pdf
[PDF; 850,1 KB]

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.

<b>Sensitive isolation of recombinant fusion protein with μMACS™ Anti-HA MicroBeads. </b>10<sup>7</sup> 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.

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.