Overview
- Product nameAnti-GFP antibodySee all GFP primary antibodies ...
- DescriptionRabbit polyclonal to GFP - ChIP Grade
- SpecificityThis antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP and EGFP. It was also shown to work with AcGFP. For WB, IP-WB and ICC/IF it is recommended that a control experiment is performed using just the permeabilised cells and the relevant secondary antibody to ensure the secondary is not masking the activity of ab290.
- Tested applicationsFlow Cyt, ELISA, ICC/IF, ChIP, IHC-FrFl, ChIP/Chip, Electron Microscopy, IHC-FoFr, ICC, IHC-P, IHC-Fr, IP, WB more details
- Immunogen
Highly purified recombinant full length protein made in Escherichia coli. The antibody is directed against the entire GFP molecule.
- Positive controlDetects 5ng of recombinant GFP (using ECL or ECL Plus) in under one minute of exposure to film.
- General notesThe 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).
Properties
- FormLiquid
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.05% Sodium azide
Constituent: 1.25% Sodium chloride - PurityWhole antiserum
- Purification notesThis antibody is provided as whole antiserum. It is not possible to determine the exact antibody concentration, since whole serum contains many other host serum proteins besides the antibody of interest.
- Primary antibody notes 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).
- Clonality Polyclonal
- IsotypeIgG
- Research Areas
Applications
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.
| Application | Notes |
|---|---|
| Flow Cyt | Flow Cyt: Use at an assay dependent dilution. |
| ELISA | ELISA: Use at an assay dependent dilution. |
| ICC/IF | ICC/IF: Use at an assay dependent dilution. |
| ChIP | ChIP: Use at an assay dependent dilution. |
| IHC-FrFl | IHC-FrFl: Use at an assay dependent dilution. |
| ChIP/Chip | ChIP/Chip: Use at an assay dependent dilution. PubMed: 17289569 |
| Electron Microscopy | EM: 1/1000 - 1/4000. |
| IHC-FoFr | IHC-FoFr: 1/200 - 1/500. |
| ICC | ICC: 1/200 - 1/1000. |
| IHC-P | IHC-P: 1/500. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol. |
| IHC-Fr | IHC-Fr: Use at an assay dependent dilution. Reported to work at dilutions up to 1/3000. |
| IP | IP: Use at an assay dependent dilution. Use at 1µl per 10cm tissue culture dish (use 10µl protein A agarose CL4B to precipitate the immune complex). |
| WB | 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. |
Target
- RelevanceGreen fluorescent protein (GFP) is a 27 kDa protein derived from the jellyfish Aequorea victoria, which emits green light (emission peak at a wavelength of 509 nm) when excited by blue light (excitation peak at a wavelength of 395 nm). GFP has become an invaluable tool in cell biology research, since its intrinsic fluorescence can be visualized in living cells. GFP fluorescence is stable under fixation conditions and suitable for a variety of applications. GFP has been widely used as a reporter for gene expression, enabling researchers to visualize and localize GFP-tagged proteins within living cells without the need for chemical staining. Other applications of GFP include assessment of protein protein interactions through the yeast two hybrid system and measurement of distance between proteins through fluorescence energy transfer (FRET) protocols. GFP technology has considerably contributed to a greater understanding of cellular physiology. YFP differs from GFP due to a mutation at T203Y; antibodies raised against full-length GFP should also detect YFP and other variants.
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Alternative names
- GFP antibodyGreen Fluorescent Protein antibody
Anti-GFP antibody images
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GFP antibody (ab290)The antibody 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 (Jackson Immuno) 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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Immunohistochemistry (Frozen sections) - GFP antibody (ab290)This image is courtesy of an Abreview submitted by Miss Danielle Harlowab290 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. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GFP antibody (ab290)This image is courtesy of an anonymous Abreviewab290 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.
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Immunocytochemistry/ Immunofluorescence - Anti-GFP antibody (ab290)This image is a courtesy of Anonymous Abreviewab290 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. An Alexa Fluor®555-conjugated goat polyclonal to rabbit IgG (H&L) 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).
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Western blot - Anti-GFP antibody (ab290)Anti-GFP antibody (ab290) at 1/10000 dilution + Lysate prepared from rabbit reticulocytes at 3 µg
Secondary
HRP-conjugated donkey monoclonal to rabbit IgG at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Observed band size : 27 kDa (why is the actual band size different from the predicted?)
Additional bands at : 60 kDa. We are unsure as to the identity of these extra bands.
Exposure time : 5 secondsThis image is a courtesy of Anonymous Abreview
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Western blot - Anti-GFP antibody (ab290)All lanes : Anti-GFP antibody (ab290) at 1/30000 dilution
Lane 1 : GFP tagged protein transfected HEK293 cell lysates
Lane 2 : GFP protein alone
Lysates/proteins at 25 µg per lane.
Secondary
HRP-conjugated Goat polyclonal to rabbit IgG at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Observed band size : 24,64 kDa (why is the actual band size different from the predicted?)
Exposure time : 3 minutesThe image is a courtesy of an abreview submitted by Vladimir Milenkovic.
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Immunoprecipitation - GFP antibody (ab290)This image is courtesy of an Abreview submitted by Vladimir Milenkovicab290 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 -
Western blotAnti-GFP antibody (ab290) at 1/2500 dilution +GFP protein (Active) (ab84191) at 0.01 µg
Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed (ab97080) at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Exposure time : 30 seconds -
Immunocytochemistry/ Immunofluorescence - GFP antibody (ab290)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
References for Anti-GFP antibody (ab290)
This product has been referenced in:
- Mittag J et al. Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions. J Clin Invest 123:509-16 (2013). IHC-P ; Mouse . Read more (PubMed: 23257356) »
- Camp ND et al. Wilms tumor gene on the X chromosome (WTX) inhibits the degradation of NRF2 through competitive binding to KEAP1. J Biol Chem : (2012). WB . Read more (PubMed: 22215675) »
