Isolation and analysis of c-myc-tagged proteins ( #130-094-251 #130-091-123 #130-091-284 #130-092-472 #130-092-471 #130-092-113 #130-094-250 )

Isolation and analysis of c-myc-tagged proteins

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

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

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.

<b>Specific isolation of recombinant fusion protein with μMACS™ Anti-c-myc MicroBeads.</b> Cells were transfected with a vector encoding c-myc-BDCA-2 and <i>in vivo</i> 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.

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.

<b>Immunofluorescence analysis of c-myc-BDCA-2 expressing CHO cells.</b> CHO cells were transiently transfected with a vector encoding c-myc-BDCA-2 and analysed by fluorescence microscopy after 24 hours of expression. <b>A</b> Detection with Anti-c-myc-FITC and CD303 (BDCA-2)-Biotin/Anti-Biotin-Alexa 546. <b>B</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.

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).

<b>Sensitive detection of recombinant fusion protein with Anti-c-myc-HRP antibody. </b>293 HEK cells were transfected with a vector encoding c-myc-BDCA-2. 5×10<sup>6</sup> 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).