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Recombinant full length protein corresponding to GFP.
The total IgG concentration has been determined to be 5 mg/ml. The specific IgG concentration is unknown. This product should be kept refrigerated at all times whilst in short term storage. Using sterilised equipment will reduce the risk of bacterial contamination.
ab290 is a highly versatile antibody that gives a stronger signal than other anti-GFP antibodies available. On Western blot the antibody detects the GFP fraction from cell extracts expressing recombinant GFP fusion proteins and has also been shown to be useful on mouse sections fixed with formalin. In Immunocytochemistry, the antibody gives a very good signal on recombinant YES-GFP chimeras expressed in COS cells (McCabe et al. 1999 and figure below). It is routinely used in Immunoprecipitation (IP) and IP-Western protocols and has been used successfully in HRP Immunohistochemistry at 1:200 on whole-mount mouse embryos. ab6556 is the purified version of this antibody (see Related Products).
This anti-GFP antibody recognizes the enhanced form of GFP as well.
Recognises EYFP (Yellow Fluorescent Protein (YFP), a genetic mutant of green fluorescent protein (GFP).
ab290 also detects venus YFP.
Our Abpromise guarantee covers the use of ab290 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
|ICC/IF||1/500 - 1/1000.|
|ChIP||Use at an assay dependent concentration.|
|IHC-FrFl||Use at an assay dependent concentration.|
|ChIP/Chip||Use at an assay dependent concentration. PubMed: 17289569|
|Electron Microscopy||1/1000 - 1/4000.|
|IHC-FoFr||1/200 - 1/500.|
|ICC||1/200 - 1/1000.|
|IHC-P||1/500 - 1/1000. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.|
|IHC-Fr||Use at an assay dependent concentration. Reported to work at dilutions up to 1/3000.|
|IP||Use at an assay dependent concentration. Use at 1µl per 10cm tissue culture dish (use 10µl protein A agarose CL4B to precipitate the immune complex).|
|WB||1/1000 - 1/2500.
It is recommended to use 12.5% SDS-PAGE and to transfer to PVDF membrane. Use 1x Blotto (or 3% BSA in PBS) for diluting and blocking. Use PBS in 3x 5min washing steps throughout the immunolabelling. Probe with ab290 at 1:1000 - 1:5000 dilution and use anti-rabbit-HRP secondary ab at 1:5000 dilution with ECL detection method. ab290 has been reported to work at 1:50,000 and dilutions around this range should be tested if high background is seen. Both incubation steps should be for 1hr at 22°C.
ab290 staining GFP in Mouse fibroblast cell by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with Triton X-100 and blocked with 3% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/1000 in BSA) for 12 hours at 4°C. A Oregon Green®-conjugated Goat anti-rabbit IgG polyclonal (1/1000) was used as the secondary antibody.
ab290 staining GFP in human HEK293 cells by Immunocytochemistry/ Imunofluorescence. Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton ×100 in PBS and blocking in 3% BSA and 2% Goat Serum was performed at 220C for 1 hour. Samples were incubated with primary antibody (1/1000: in 3% BSA, 2% Goat Serum in 0.1%Triton X100 PBS) for 12 hours at 4°C. A goat anti-rabbit Alexa Fluor®555 ab150078) was used as secondary antibody. In the figure, ab290 antibody specifically binds to the HEK293 cells expressing GFP. The non-GFP-expressing cells are not recognized by ab290 (pointed with white arrow).
ab290 staining GFP in GFP-transfected NIH3T3 cells. The cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA / 0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab290 at 1/200 dilution overnight at +4°C followed by incubation with ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488), for 1 hour, at 1μg/ml.
Under identical experimental conditions, when compared to the basal level of GFP expression in transfected NIH3T3 cells, the cells upon which ab290 was applied gave a stronger signal in the 488 channel, indicating that ab290 is binding to GFP and therefore eliciting signal amplification.
ab290 was also applied to non-GFP-transfected NIH3T3 cells, which produced no positive staining, indicating specificity for GFP. Nuclear DNA was labelled with 1.43μM DAPI (blue).
Immunofluorescence images showing similar localization of Yes-GFP (first 10 aa's of Yes PTK fused to the N-terminus of GFP) to full length Yes PTK. A: Distribution of Yes detected using mouse anti-Yes Ab followed by Texas Red-conjugated anti-mouse Ab. B: Chimeric GFP's detected using rabbit anti-GFP Ab (Abcam ab290) followed by FITC-conjugated anti-rabbit Ab.
Image kindly provided by L.G. Berthiaume. Taken from J. McCabe and L.G. Berthiaume, Functional Roles for Fatty Acylated Amino-terminal Domains in Subcellular Localization, Molecular Biology of the Cell 10:3771-3786, 1999
Flow cytometry analysis of paraformaldehyde-fixed human differentiated hNSCs cells prepared through accutase, quantifying GFP with ab290 diluted 1/300 incubated for 20 hours at 25°C. Permeableization was through 0.25% Triton X-100 in DPBS. Secondary was a polyclonal goat anti-rabbit Alexa Fluor® 488 at 1/500. The gating strategy was against undifferentiated stem cells (shown in white).
ab290 Immunoprecipitate in human HEK 293 cells transfected with Annexin1-GFP. 25µg of cell lysate incubated with primary antibody and matrix (Protein G) in 1% TX-100, 10% glycerol, 1X PBS for 16 hours at 4°C. For Western blotting an HRP conjugated HRP goat polyclonal to rabbit Ig was used at a dilution at 1:5000. Line 1: Lysate of HEK 293 cells expressing Annexin1-GFP fusion Line 2: IP with anti GFP Ab Line 3: Not bound fraction
This image is a courtesy of Anonymous Abreview
The image is a courtesy of an abreview submitted by Vladimir Milenkovic.
Primary diluted in 1XPBST, 5% milk and incubated with sample for 16 hour at 4°C.
ab290 at a 1/2000 dilution staining GFP-labelled nerve fibres from Axolotls (Ambystoma mexicanum). The tissue sections were paraformaldehyde fixed and blocked with serum prior to incubation with the antibody for 24 hours. Bound antibody was detected using a biotinylated goat anti-rabbit polyclonal antibody.
ab290 staining dog hearts (Adv-GFP injection) tissue sections by IHC-P. Sections were PFA fixed and subjected to heat mediated antigen retrieval in citric acid (Ph6.0, 0.05% Tween20) prior to blocking with 10% serum for 30 mins at 37°C. The primary antibody was diluted 1/1000 in PBS and incubated with the sample for 1 hour at 25°C. A HRP-conjugated goat anti-rabbit IgG was used as the secondary antibody.
ab290 was used to detect transplanted bone marrow derived cells in paraffin embedded mouse brain tissue. The image was taken at 40X magnification. The GFP290 antibody(red) was visualized with a Cy3 anti-rabbit and the nuclei (blue) have been counterstained with bisbenzimide (Hoechst Stain). The image illustrates vascular association. The picture was kindly given to Abcam by the authors of the reference Hess, D.C et al. Bone Marrow as a Source of Endothelial Cells and NeuN-Expressing Cells After Stroke. Stroke 33(5) pp 1362-1368 (2002).
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