Isolation and analysis of His-tagged proteins
Background The polyhistidine (His) tag is an amino acid motif that consist of six histidine residues which are added at the C-terminus or N-terminus of recombinant protein for affinity purification. High-quality monoclonal anti-His antibodies are offered by Miltenyi Biotec either coupled to µMACS™ MicroBeads for specific isolation of His-tagged proteins on an analytical scale or conjugated to biotin, FITC, or horseradish peroxidase (HRP) for analysis of recombinant His-tagged protein. Isolation and detection of His-tagged proteins Isolation of His-tagged proteins The µMACS™ and MultiMACS™ His Isolation Kits take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure His-tagged proteins. The advantages of MACS Technology for protein isolation are: Specificity – high-quality monoclonal antibodies coupled to MACS MicroBeads assure specific isolation of His-tagged proteins with low background. Sensitivity – fast reaction kinetics of tiny (50 nm), non-sedimenting MACS MicroBeads allow sensitive isolation even when working with rare proteins. Speed – less than 2 hours to high-purity His-tagged proteins! The µMACS His Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator. The procedure can easily be upscaled with the MultiMACS His Isolation Kits to a semi- or fully-automated, high-throughput processing of up to 96 samples in parallel by utilizing the MultiMACS 96 Separator. For medium- to large-scale purification of His-tagged proteins the ProCatch His Resin is recommended (fig. 1). Detection of His-tagged proteins The monoclonal Anti-His antibody is available with various conjugates. The fluorochrome-conjugated (FITC or PE) Anti-His antibodies enable an immunofluorescence analysis for example, by fluorescence microscopy or FACS (fig. 2). Various detection methods are applicable using the biotinylated Anti-His antibody in combination with appropriate streptavidin conjugates. For specific and sensitive detection of His-tagged proteins by Western blot or ELISA analysis the Anti-His-HRP and the Anti-His-HRP (C-term.) are available. Directly coupled to horseradish peroxidase (HRP), both antibodies simplifiy these immunological assays as incubation with secondary antibodies is not necessary. The Anti-His-HRP (C-term.) shows even higher sensitivity and specificity for C-terminal His-tagged proteins than the Anti-His-HRP. The latter detects both N- and C-terminal His-tagged proteins. Antibody specifications Target: His epitope (HHHHHH) Isotype Anti-His antibody: mouse IgG2b Isotype Anti-His antibody (C-terminal): 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 isolation flyer.pdf [PDF; 814,4 KB]		Figure 1 Purification of a 30 kDa histidine-tagged protein from prokaryotic cell lysate with ProCatch His Resin. Marker (lane 1), lysate from bacterial extract (2), the flow-through (3), and the eluate (4) were separated by SDS-PAGE and Coomassie stained. Figure 2 Intracellular labeling and FACS analysis of mPD1-His expressing CHO cells. CHO cells were transiently transfected with a mPD1-His encoding vector and analysed by flow cytometry. CHO cells were fixed, permeabilized and labeled with Anti-His-FITC (B) or Mouse IgG1-FITC isotype control (A), respectively. A B

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