Overview

  • Product nameAnti-GFP antibody - ChIP GradeSee all GFP primary antibodies ...
  • Description
    Rabbit polyclonal to GFP - ChIP Grade
  • SpecificityRecognises EYFP (Yellow Fluorescent Protein (YFP), a variant of green fluorescent protein (GFP)
  • 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 control
    • Detects 5ng of recombinant GFP (using ECL or ECL Plus) in under one minute of exposure to film. Human HEK293 cells, rabbit reticulocytes
  • General notes

    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).

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.
  • 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 Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
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. 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.

Target

  • RelevanceFunction: Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca2+ -activated photoprotein aequorin.

    Subunit structure: Monomer.

    Tissue specificity: Photocytes.

    Post-translational modification: Contains a chromophore consisting of modified amino acid residues. The chromophore is formed by autocatalytic backbone condensation between Ser-65 and Gly-67, and oxidation of Tyr-66 to didehydrotyrosine. Maturation of the chromophore requires nothing other than molecular oxygen.

    Biotechnological use: Green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors. Fluorescent proteins and its mutated allelic forms, blue, cyan and yellow have become a useful and ubiquitous tool for making chimeric proteins, where they function as a fluorescent protein tag. Typically they tolerate N- and C-terminal fusion to a broad variety of proteins. They have been expressed in most known cell types and are used as a noninvasive fluorescent marker in living cells and organisms. They enable a wide range of applications where they have functioned as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions. Can also be used as a molecular thermometer, allowing accurate temperature measurements in fluids. The measurement process relies on the detection of the blinking of GFP using fluorescence correlation spectroscopy.

    Sequence similarities: Belongs to the GFP family.

    Biophysicochemical properties: Absorption: Abs(max)=395 nm
    Exhibits a smaller absorbance peak at 470 nm. The fluorescence emission spectrum peaks at 509 nm with a shoulder at 540 nm.
  • Alternative names
    • GFP antibody
    • Green Fluorescent Protein antibody
    • yfp antibody

Anti-GFP antibody - ChIP Grade images

  • 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

  • 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. An Alexa Fluor®555-conjugated goat polyclonal to rabbit IgG (H&L) 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).

    See Abreview

  • 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

    See Abreview

  • Anti-GFP antibody - ChIP Grade (ab290) at 1/2500 dilution + Active A. victoria GFP full length protein (ab84191) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) 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?)


    Exposure time : 30 seconds
  • Anti-GFP antibody - ChIP Grade (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?)


    Exposure time : 5 seconds

    This image is a courtesy of Anonymous Abreview

    See Abreview

  • All lanes : Anti-GFP antibody - ChIP Grade (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 minutes

    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.

    See Abreview

  • 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.

    See Abreview

  • 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.

    See Abreview

  • 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).

References for Anti-GFP antibody - ChIP Grade (ab290)

This product has been referenced in:
  • Dawson MA  et al. Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia. Leukemia 28:311-20 (2014). Read more (PubMed: 24220271) »
  • Bettum IJ  et al. Metastasis-associated protein S100A4 induces a network of inflammatory cytokines that activate stromal cells to acquire pro-tumorigenic properties. Cancer Lett 344:28-39 (2014). Read more (PubMed: 24215866) »

See all 409 Publications for this product

Product Wall

Abreviews
Application Western blot
Sample Mouse Recombinant protein (HEK293T cells over expressing mouse 5-HT6R)
Loading amount 50 µg
Specification HEK293T cells over expressing mouse 5-HT6R
Gel Running Conditions Reduced Denaturing (8% Tris-Glycine gel)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 22°C
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Abcam user community

Verified customer

Submitted Mar 21 2013

For antibodies to have optimal activity, we suggest that it is best that they are kept at -20°C or -80°C in small aliquots and to only have a small volume kept at 4°C.

We urge all our customers to follow the storage information st...

Read More

We do not have this specific antibody linked to anything for IP, however we do have the GFP antibody ab69314, which is linked to Sepharose beads. This should work since this product is a purified antibody, while ab290 is from serum.

I hope thi...

Read More
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Colon)
Specification Colon
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: 10 mM Citrate
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Feb 12 2013

We haven't tested this ourselves, however, as the antibody is directed against the recombinant full length protein and is polyclonal, I am quite positive that this antibody will recognize the GFP variant.

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Sample Fruit fly (Drosophila melanogaster) Cell (drosophila adult brain wholemount)
Specification drosophila adult brain wholemount
Permeabilization Yes - pbst
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 23 2013


There have been several customers who have used this antibody successfully in combination with samples fixed with other fixatives other than PFA. Here are three Abreviews were methanol has been used:

http://www.abcam.com/GFP-antibody-ab29...

Read More

To our knowledge the detection of turbo-GFP has not been tested with ab290. Turbo-GFP is an improved variant of the green fluorescent protein CopGFP cloned from copepod Pontellina plumata , which is a different organism that the classical/original GFP ...

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Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (U2oS)
Specification U2oS
Fixative Paraformaldehyde
Permeabilization Yes - 0.1% Tween
Blocking step BSA as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 22°C
Username

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Verified customer

Submitted Aug 06 2012

Application Western blot
Loading amount 25 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Mouse Tissue lysate - whole (spinal cord)
Specification spinal cord
Treatment co-infected with adeno virus either expressing Cre recombinase or flox-STOP-flox-GFP
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

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Verified customer

Submitted Mar 31 2014

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"