| Background |
The non-toxic green fluorescent protein (GFP) is a 26.9 kDa protein originally cloned from the jellyfish. GFP absorbs blue light and emits green light with no need for exogenous substrates and cofactors. Thus, several derivates of GFP have been developed to be used as a reporter to monitor gene expression and protein localization in vivo.
A high-quality monoclonal Anti-GFP antibody is offered by Miltenyi Biotec that recognizes all GFP derivates and is available either coupled to µMACS™ MicroBeads for specific isolation of GFP-tagged proteins or conjugated to horseradish peroxidase (HRP) for appropriate analysis of GFP fusion proteins. |
| Isolation and detection of GFP-tagged proteins |
Isolation of GFP-tagged proteins
The µMACS™ and MultiMACS™ GFP Isolation Kit take advantage of the well-established MACS® Technology; thus, allows fast and specific isolation of pure GFP-tagged proteins. Purified GFP fusion proteins have successfully been applied for several different downstream experiments including mass spectrometry analysis2,6. The advantages of MACS Technology for protein isolation are:
Specificity – high-quality monoclonal antibodies coupled to MACS MicroBeads assure specific isolation of recombinant proteins fused to GFP or one of its derivates.
Sensitivity – fast reaction kinetics of the tiny (50 nm), non-sedimenting MACS MicroBeads allow sensitive isolation even when working with rare proteins.
Speed – less than 2 hours to high-purity GFP-tagged protein!
The µMACS GFP Isolation Kit was developed for manual, low-throughput applications with the µMACS Separator.
The procedure can easily be upscaled with the MultiMACS GFP 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 GFP-tagged proteins
The monoclonal Anti-GFP-HRP antibody allows specific and sensitive detection of GFP fusion proteins (fig. 1). Directly coupled to horseradish peroxidase (HRP), the antibody simplifies Western blot or ELISA analysis as incubation with secondary antibodies are not necessary.
Antibody specifcations
Target: whole green fluorescent protein (238-residue polypeptide)
Tag origin: Aequoria victoria jellyfish
Isotype: mouse IgG1
Specificity: enhanced GFP (EGFP), blue fluorescent protein (EBFP), cyan fluorescent protein (ECFP), and yellow fluorescent protein (EYFP). |
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| Figure |
| Sensitive detection of recombinant GFP fusion proteins. 20 ng, 10 ng, 5 ng, and 1 ng GFP fusion protein were separated by SDS-PAGE, blotted on a PVDF membrane and protein bands were detected with Anti-GFP-HRP (1:5,000, 1 h, room temperature) and ECL detection reagents (GE Healthcare). |
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