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Recombinant full length protein corresponding to GFP aa 1-246.
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GKLPVPWPTL VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRA EVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGIT HGMDELYK
Designed to detect GFP and its variants in ELISA (sandwich or capture), immunoblotting and immunoprecipitation. Fluorescein conjugated anti-GFP was assayed by immunofluorescence microscopy on prokaryotic (E.coli) and eukaryotic (CHO cells) expression systems and was shown to detect GFP containing inserts. Significant amplification of signal was detected using fluorochrome conjugated anti-GFP relative to the fluorescence of GFP alone.
In case of unexpected background, use pre-adsorbed secondary antibodies.
Fluorescein isothiocyanate (FITC) (MW 390 daltons) Absorption Wavelength: 495 nm Emission Wavelength: 528 nm Fluorochrome/Protein Ratio: 3.5 moles FITC per mole of Goat IgG
Our Abpromise guarantee covers the use of ab6662 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-Fr||Use at an assay dependent concentration.|
|WB||1/10000 - 1/100000.|
|ICC/IF||1/200 - 1/400.|
|IF||1/500 - 1/2500.|
Immunofluorescence Microscopy using ab6658. Tissue: Drosophila melanogaster late stage embryonic central nervous system. Fixation: 0.5% PFA. Antigen retrieval: not required. Primary antibody: Anti-GFP antibody at a 1/1,000 for 1 h at RT. Secondary antibody: AlexaFluor 488™ conjugated anti-Goat antibody at 1/300 for 45 min at RT. Panel A: shows a lateral view (ventral left). Panels B and C: shows ventral views of whole mount embryos at 63x magnification (plus 2x digital zoom). In all panels, anterior is up. Staining: tau-GFP cell bodies (large arrowhead) and axons of motorneurons (arrow) and interneurons (small arrowhead) as green fluorescent signal.
Western Blot of ab6662. Load: 50 ng per lane. Secondary antibody: Fluorescein goat secondary antibody at 1/1,000 for 60 min at RT. Block: MB-070 for 30 min at
ab6662 staining mouse brain tissue sections (inducible GFP reporter) by IHC-Fr. The tissue was paraformaldehyde fixed and blocked with serum and then incubated with the antibody at a 1/1000 dilution for 1 hour.
Staining is shown in the left hand panel. The middle panel shows staining with a rabbit anti-GFP antibody and the right hand panel shows the merged images (plus DAPI). ab6662 gives no noticable background and it is found that when viewing on an epifluorescent the exposure time is significantly reduced.
Immunofluorescence Microscopy using ab6658. Tissue: Sf-1:Cre mice crossed to the Z/EG reporter line. Mouse brain (coronal view, 20X magnification). Fixation: 4%PFA/PBS with o/n fixation, and subsequently transferred to a 30% sucrose solution.Antigen retrieval: frozen in OCT freezing medium (Sakura) and cryostat sectioned at 40 microns. Primary antibody: Goat anti- GFP was used at 1/500 dilution in free floating imunnohistochemistry to detect GFP. Secondary antibody: Fluorchrome conjugated Anti-goat IgG secondary antibody was used for detection at 1:500 at 1/10,000 for 45 min at RT.Localization: Sf-1+ neurons and their processes of the ventromedial nucleus of the hypothalamus. Staining: eGFP as green fluorescent signal and sections were counterstained with DAPI.
These pictures show confocal immunofluorescence using GFP-expressing glial cells (green) transplanted into the lesioned rat spinal cord. This was detected using ab6662 and a standard FITC filter set. Axons are labelled red by an antibody to neurofilament-200 and a rhodamine secondary antibody. The upper panel shows the centre of the transplant site at low power. Numerous GFP-positive cells can be seen mingling with axons. The lower panel shows, at high power in a single optical section, how ab6662 reveals the morphology of the transplanted cells to such an extent that their close interactions with axons are obvious - the cell depicted can be seen wrapping around a neurofilament-200 positive axon.
These images were kindly supplied as part of the review submitted by Andrew Toft.
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