| Background |
The c-myc peptide (EQKLISEEDL) originates from the human c-myc proto-oncogene, which is overexpressed in a wide range of human cancers. It has become a popular fusion tag for mammalian recombinant protein expression.
A high-quality monoclonal Anti-c-myc antibodies is offered by Miltenyi Biotec either coupled to µMACS™ MicroBeads for specific isolation of c-myc-tagged proteins or conjugated to biotin, FITC, or horseradish peroxidase (HRP) for analysis of the recombinant c-myc-tagged protein. |
| Isolation and detection of c-myc-tagged proteins |
Isolation of c-myc-tagged proteins
The µMACS™ and MultiMACS™ c-myc Isolation Kits take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure c-myc-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 c-myc-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 c-myc-tagged proteins!
The µMACS c-myc Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator.
The procedure can easily be upscaled with the MultiMACS c-myc 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 c-myc-tagged proteins
The monoclonal Anti-c-myc antibody is available with various conjugates:
The FITC-conjugated Anti-c-myc antibody enables an immunofluorescence analysis, e.g. by fluorescence microscopy (fig. 2). Directly coupled to horseradish peroxidase (HRP), the Anti-c-myc-HRP antibody simplifies immunological methods, as incubation with secondary antibodies is not necessary. The Anti-c-myc-HRP antibody was generated for specific and sensitive detection of c-myc-tagged proteins by Western Blot (fig. 3) or ELISA analysis. The biotinylated Anti-c-myc antibody can be used for various detection methods in combination with appropriate streptavidin conjugates.
Target sequence: EQKLISEEDL
Tag origin: Human c-myc protooncogene
Isotype: mouse IgG1 |
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| Figure 1 |
| Specific isolation of recombinant fusion protein with μMACS™ Anti-c-myc MicroBeads. Cells were transfected with a vector encoding c-myc-BDCA-2 and in vivo labeled with 35S-methionine. The purification of recombinant c-myc-BDCA-2 protein was performed using Anti-c-myc MicroBeads and, as a negative control, Anti-HA MicroBeads. The figure shows an autoradiogram after SDS-PAGE. |
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| Figure 2 |
| Immunofluorescence analysis of c-myc-BDCA-2 expressing CHO cells. CHO cells were transiently transfected with a vector encoding c-myc-BDCA-2 and analysed by fluorescence microscopy after 24 hours of expression. A Detection with Anti-c-myc-FITC and CD303 (BDCA-2)-Biotin/Anti-Biotin-Alexa 546. B Detection with Anti-c-myc-Biotin/Anti-Biotin-Alexa 546 and CD303 (BDCA-2)-FITC; red: Alexa 546-labeling, green: FITC-labeling, yellow: FITC-Alexa 546 double-labeling. |
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| Figure 3 |
| Sensitive detection of recombinant fusion protein with Anti-c-myc-HRP antibody. 293 HEK cells were transfected with a vector encoding c-myc-BDCA-2. 5×106 cells were lysed and recombinant protein c-myc-BDCA-2 was purified using the μMACS™ Anti-c-myc Isolation Kit. 1/100, 1/500, and 1/1000 of the eluate was separated by SDS-PAGE, blotted on a PVDF membrane and detected using Anti-c-myc-HRP (1:10,000, 1 h, room temperature) and ECL reagents (GE Healthcare). |
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