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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GFP antibody (ab290)
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Immunohistochemistry (Frozen sections) - GFP antibody (ab290)
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GFP antibody (ab290)
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Product Name
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GFP antibody
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See all GFP antibodies (26)...
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Product type
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Primary antibodies
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Description
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Rabbit polyclonal to GFP - ChIP Grade
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Immunogen
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Highly purified recombinant full length protein made in Escherichia coli. The antibody is directed against the entire GFP molecule.
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Specificity
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This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP and EGFP.
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Tested applications
(see key)
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ChIP, ChIP/Chip, ELISA, EM, Flow Cyt, ICC/IF, IHC-FoFr, IHC-Fr, IHC-P, IP, WB
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Abreviews
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Application notes
(see key)
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Recommended dilutions
Electron Microscopy: 1/100 - 1/4000.
ICC / IF: 1/200 - 1/2000. Cells should be analysed 16-24hr following transfection to allow for GFP chromophore development. Cells should be washed in PBS, fixed in 4% paraformaldehyde in PBS, pH 7.4, for 20min and permeabilized with 0.1% Triton X-100 in PBS for 1min at room temperature. Blocking should be performed with normal donkey serum in PBS. Antibodies should be diluted in 4% normal donkey serum (the secondary Ab we use is raised in donkey) to prevent non-specific binding. To detect the Golgi apparatus in living transfected cells, BODIPY TR-ceramide (1.5µM) should be added for 0.5-2hr before viewing. Living transfected cells should be incubated with DiI-LDL (1µg/ml) for 1hr prior to fixation. This allows incorporation of the fluorescent lipoprotein particle into endosomes. For live cell fluorescence analysis coverslips should be initially washed with pre-warmed PBS and mounted onto glass slides in PBS using vacuum grease or nail polish as a sealant.
IHC-F: Use at an assay dependent dilution. ab290 has been reported to work at dilutions up to 1/3000. For blocking use normal donkey serum (where the secondary is raised in donkey) in PBS and then incubate with ab290 at 37°C in high humidity.
IHC-P: 1/200 - 1/500, although ab290 has been reported to work at dilutions up to 1/2000. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol. For blocking use normal donkey serum (where the secondary is raised in donkey) in PBS and then incubate with ab290 at 37°C in high humidity.
IHC-Fr: Use at an assay dependent dilution. For blocking use normal donkey serum (where the secondary is raised in donkey)in PBS and then incubate with ab290 at 37°C in high humidity.
IP: Use at 1µl per 10cm tissue culture dish (use 10µl protein A agarose CL4B to precipitate the immune complex).
WB: 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 antibody at 1/5000 dilution with ECL detection method. ab290 has been reported to work at dilutions of 1/50,000 and dilutions around this range should be tested if high background is observed. Both incubation steps should be for 1hr at 22°C. The antibody detects a band at approximately 27 kDa.
ChIP: Use at an assay dependent dilution.
ChIP on chip: Use at an assay dependent dilution (from PubMed:17289569).
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.
Not tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
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Positive control
(see definition)
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Detects 5ng of recombinant GFP (using ECL or ECL Plus) in under one minute of exposure to film.
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Research areas
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Tags & Cell Markers >> Fusion / Marker Proteins >> GFP
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Relevance
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Green fluorescence protein (GFP) is a 27 kDa protein derived from the jellyfish Aequorea victoria, which emits green light (emission peak at a wavelenth of 509 nm) when excited by blue light (excitation peak at a wavelenth of 395 nm). Green Fluorescent Protein (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 technnology 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.
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 for GFP antibody below).
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Raised in
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Rabbit
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Clonality
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Polyclonal
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Isotype
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IgG
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Purity
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Whole antiserum
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Storage buffer
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Preservative: 0.05% Sodium Azide
Material safety datasheet (MSDS) for this product:
Sodium Azide MSDS
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Purification notes
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This 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.
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Form
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Liquid
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Concentration
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Concentration not determined - why is this?
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Storage instructions
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Store at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
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Notes
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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.
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At Abcam, we have one centralized database to hold all of our product information, so that everything we know about this GFP antibody is on this datasheet. But please do contact us if you would like any reassurance! |
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See below for GFP antibody images, references, products related to ab290 and other tools.
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GFP antibody images:
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 Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - 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)
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. This image is courtesy of an Abreview submitted by Miss Danielle Harlow
See Abreview
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - GFP antibody (ab290)
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. This image is courtesy of an anonymous Abreview
See Abreview
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Immunocytochemistry/ Immunofluorescence - GFP antibody (ab290)
ab290 staining GFP in HEK293 cells by Immunocytochemistry/ immunoflurescence. Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton ×100 in PBS and blocking in a blocking solution containing 3% BSA and 2%Goat Serum was done at 220C for 1 hour. Samples were incubated with primary antibody (1/1000: in dilution buffer containing 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, diluted 1/1000, 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). This image is a courtesy of Anonimous Abreview
See Abreview
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Western blot - GFP antibody (ab290)
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 seconds This image is a courtesy of Anonimous Abreview
See Abreview
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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
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References for GFP antibody (ab290)
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This product has been used in: (two most recent references)
Thorsen TS et al. Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD. Proc Natl Acad Sci U S A 107:413-8 (2010). IP. PubMed: 20018661
Kennedy MW et al. A co-dependent requirement of xBcl9 and Pygopus for embryonic body axis development in Xenopus. Dev Dyn 239:271-83 (2010). WB. PubMed: 19877304
See all 235 publication references for this product.
If you publish research using ab290 please let us know so that we can cite the reference on this datasheet.
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Search PubMed (MEDLINE) for references to GFP
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GFP antibody - more information
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Customer reviews (feedback) regarding GFP antibody |
Customer FAQs regarding GFP antibody |
Protocols for GFP antibody |
Price and availability of products related to GFP antibody |
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GFP antibody - other tools:
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Contact Abcam with a Technical Enquiry about GFP antibody |
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