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Rabbit Polyclonal to GFP / Green Fluorescent Protein

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Background

Green fluorescence protein (GFP) is a 27 KDa protein derived from the bioluminiscent jellyfish Aquorea victoria, emiting green light (λ=509 nm) when excited (excitation by Blue or UV light, absorption peak λ=395 nm).

GFP is a useful tool in cell biology research, as its intrinsic fluorescence can be visualized in living cells. Light-stimulated GFP fluorescence is species-independent and a fluorescence has been reported from many different types of GFP-expressing hosts, including microbes, invertebrates, vertebrates and plants. No exogenous substrates and cofactors are required for the fluorescence of GFP, since GFP autocatalytically forms a fluorescent pigment from natural amino acids present in the nascent protein.

GFP fluorescence is stable under fixation conditions and suitable for a variety of applications. GFP is widely used as a reporter (tag) for gene expression, enabling researchers to visualize and localize GFP-tagged proteins within living cells without any further staining. Other applications of GFP include measurement of distance between proteins through fluorescence energy transfer (FRET) protocols. To increase a fluorescence intensity of GFP, chomophore mutations have been created. The EnhancedGFP has a fluorescence 35 times more intense than the wt-GFP. Mutagenesis of GFP has produced also many mutants (e.g. Yellow Fluorescent Protein, Cyan Fluorescent Protein) with warying spectral properties. Antibodies raised against full-length GFP variants should also detect other variants of the protein.

Specificity

The polyclonal antibody recognizes GFP, EGFP, EYFP fusion proteins in all species.

Regulatory Status

Immunogen

EGFP, a native full-length protein

Species Reactivity:

  • Recognizes fusion proteins in all species

Negative Species:

Applications:

  • Immunoprecipitation
    Recommended dilution:

    10-20 μg/sample
    Application note

  • Western Blotting
    Recommended dilution:

    0.5-1.5 μg/ml
    Positive control:

    transfected cells

    Negative control:

    non-transfected cells

  • Immunocytochemistry
    Recommended dilution:

    Purified Antibody: 1-3 μg/ml

    Positive control:

    transfected cells

    Negative control:

    non-transfected cells

Usage note:
Indicated dilutions are recommended starting points for use of this product. Working concentrations should be determined by the investigator.

Product Specific References

  • *Valenta T, Lukas J, Doubravska L, Fafilek B, Korinek V: HIC1 attenuates Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies. EMBO J. 2006 Jun 7;25(11):2326-37. Epub 2006 May 25. [Abstract]
  • For research use only. Not for drug, diagnostic or other use.

    Example Data


    GFP-confocal
    Fig. 1. Confocal microscopy



    Fig. 1. Confocal microscopy images of COS-7 cells transfected with expression constructs encoding membrane-tethered EGFP (membrane-EGFP; top) or nuclear Polycomb 2-EYFP fusion protein (Pc2-EYFP; bottom).
    The natural fluorescence of the produced proteins is shown in the green channel (left), polyclonal anti-GFP antibody signal was detected in the red channel (right). The system was carefully tested for overlap of these two optical channels and images were scanned separately in sequential scanning mode. The blue nuclear stain is also shown.







    IP_final
    Fig. 2. Immunoprecipitation



    Fig. 2. Immunoprecipitation of GFP-NLS from HEK293 cells using anti-GFP antibody.
    HEK293 cells were transfected with expression construct encoding GFP-NLS protein. Twenty hours post transfection cells were lysed in non-denaturating conditions (Lysis buffer: 20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% Triton X-100, inhibitors of proteases). Aliquots of cell lysate were immunoprecipitated using a polyclonal anti-GFP antibody (lane 2) or a pre-immune rabbit serum (lane 3). Immunoprecipitates together with a sample of the cell lysate (lane 1) were separated on SDS-PAGE polyacrylamide gel and immunoblotted with the anti-GFP antibody. The positions of molecular weight markers in kDa are indicated at the left.

    Unless indicated otherwise, all products are For Research Use Only and not for diagnostic or therapeutic use. Not for resale or transfer either as a stand-alone product or as a component of another product without written consent of EXBIO. EXBIO will not be held responsible for patent infringement or other violations that may occur with the use of our products. All orders are accepted subject to EXBIO´s term and conditions which are available at www.exbio.cz.
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