Overview

  • Product nameAnti-GFP antibody - ChIP Grade
    See all GFP primary antibodies
  • Description
    Rabbit polyclonal to GFP - ChIP Grade
  • Tested applicationsFlow Cyt, ELISA, ICC/IF, ChIP, IHC-FrFl, ChIP/Chip, Electron Microscopy, IHC-FoFr, ICC, IHC-P, IHC-Fr, IP, WBmore details
  • Immunogen

    Recombinant full length protein corresponding to GFP.

  • 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).

    ab290 also detects venus YFP.

Properties

  • FormLiquid
  • Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • 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.
  • ClonalityPolyclonal
  • 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 Abreviews Notes
Flow Cyt 1/50 - 1/300.

See Abreview section for protocols.  Also, ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

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 5 µg for 1 µg of chromatin.

See Abreviews section for protocols. Also see PubMed ID 17289569 for more information.

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.

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

  • ab290 staining GFP in U2OS cells expressing TRF2-GFP fusion protein by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with NP40 and blocked with 3% BSA for 1 hour at 21°C. Samples were incubated with the primary antibody (1/1000 in PBS + 3% BSA) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal at a dilution of 1/500 was used as the secondary antibody.

    Green - GFP.
    Blue - DAPI.

    See Abreview

  • ab290 immunoprecipitating GFP in HEK293 nuclear lysate expressing GFP. 20µg of lysate was incubated with primary antibody (1 µg/mg lysate) and matrix (Protein G) for 16 hours at 4°C in AFC low salt buffer. For western blotting ab290 (1/5000) was used to confirm successful immunoprecipation.

    Lane 1: HEK293 nuclear lysate expressing GFP input.
    Lane 2: IP of HEK293 nuclear lysate expressing GFP.
    Lane 3: Cells with no GFP.

    See Abreview

  • Anti-GFP antibody - ChIP Grade (ab290) at 1/2500 dilution + Recombinant A. victoria GFP 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

    Secondary antibody - goat anti-rabbit HRP preadsorbed (ab97080)

  • B - Frontal confocal projection through the brain showing CD8::GFP in the MB and nls.dsRED in the Kenyon cell nuclei. Some expression can be seen at lower levels in other regions of the brain. Scale bar = 200 μm.

    C- Posterior view showing CD8::GFP localized to the calyx and nls.dsRED in the Kenyon cell nuclei.

    Whole flies were fixed in PFAT/DMSO (4% paraformaldehyde in 1X PBS +0.1% Triton X-100+5% DMSO) for one hour then washed in 1X PBS. Brains were microdissected in 1X PBS then post fixed in PFAT/DMSO for 20 mins and stored in MeOH at -20°C. Following rehydration in PBT (1X PBS + 0.5% triton X-100) brains were blocked in 5% normal goat serum in PBT for two hours at room temperature. They were then incubated overnight at room temperature with the primary antibody rabbit anti-GFP (ab290, 1/20000) then incubated overnight at 4°C with secondary antibody (Alexa Fluor® 488-conjugated goat anti-rabbit IgG, 1/200) and mounted. For confocal microscopy, optical sections were taken with a Leica TCS SP5 DM6000B Confocal Microscope. Image stacks were taken at intervals of 1 μm and processed with Leica Application Suite Advanced Fluorescence (LAS AF) software.

  • 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

  • ab290 immunoprecipitate in human HEK293 cells transfected with Annexin1-GFP. 25µg of cell lysate was incubated with the 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.

    Lane 1: Lysate of HEK293 cells expressing Annexin1-GFP fusion protein.
    Lane 2: IP with anti-GFP.
    Lane 3: Not bound fraction.

    See Abreview

  • All lanes : Anti-GFP antibody - ChIP Grade (ab290) at 1/5000 dilution

    Lane 1 : LNCaP whole cell lysate - pEGFP empty vector
    Lane 2 : LNCaP whole cell lysate - pEGFP-PKD1 transfected

    Lysates/proteins at 20 µg per lane.

    Secondary
    HRP-conjugated goat anti-rabbit IgG at 1/10000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Exposure time : 10 seconds

    This image is courtesy of an anonymous Abreview

    Blocked with 5% milk for 1 hour at 23°C.

    Incubated with the primary antibody for 16 hours at 4°C.

    See Abreview

  • All lanes : Anti-GFP antibody - ChIP Grade (ab290) at 1/5000 dilution

    Lane 1 : COS7 whole cell lysate - transfected with GFP-Eml4
    Lane 2 : COS7 whole cell lysate - transfected with GFP

    Lysates/proteins at 20 µg per lane.

    Secondary
    HRP-conjugated pig anti-rabbit IgG at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 30 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 seconds

    This image is courtesy of an Abreview submitted by S Houtman

    Blocked with 5% milk for 1 hour at 20°C.

    Incubated with the primary antibody for 18 hours at 4°C in TBS containing 2% milk and 1% Tween.

    Predicted MW of Eml4 ~ 120 kDa.

    See Abreview

  • Immunohistochemistry (Free Floating) analysis of mouse brain tissue sections labelling GFP with ab290. Tissue was fixed with 4% PFA, frozen 30 µm sections were blocked for 1 hour at room temperature with 10% normal goat serum + donkey anti-mouse IgG Fab fragments (0.1 mg/ml). Sections were incubated with the primary antibody at a dilution of 1/1000 in TBS + 0.25% Triton-X for 16 hours at 4°C. A Cy2®-conjugated donkey anti-rabbit IgG (H+L) at a dilution of 1/200 was used as the secondary antibody.

    Image shows anti-NeuN (red), DAPI (blue), and anti-GFP staining of GFP-cre (green, yellow with NeuN colocalization).

    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

  • 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).

    See Abreview

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

This product has been referenced in:
  • Combs B  et al. Gene Therapy Models of Alzheimer's Disease and Other Dementias. Methods Mol Biol 1382:339-66 (2016). WB . Read more (PubMed: 26611599) »
  • Noy PJ  et al. TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions: EVIDENCE FOR DISTINCT BINDING MECHANISMS FOR DIFFERENT TspanC8 PROTEINS. J Biol Chem 291:3145-57 (2016). Read more (PubMed: 26668317) »

See all 915 Publications for this product

Product Wall

Abreviews
Abcam guarantees this product to work in the species/application used in this Abreview.
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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Verified customer

Submitted Mar 21 2013

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.

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 ...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
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
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Submitted Feb 12 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
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
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Submitted Aug 06 2012

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Kidnery GFP transgenic mouse)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: citrate buffer (pH 6)
Permeabilization Yes - Triton-x 0.1% in PBS
Specification Kidnery GFP transgenic mouse
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
Fixative Paraformaldehyde
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Submitted Aug 03 2016

Application Western blot
Sample Human Cell lysate - whole cell (Brain stem cells)
Gel Running Conditions Non-reduced Denaturing (Gradient Gel)
Loading amount 1 µg
Specification Brain stem cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
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Submitted Jul 14 2016

Application Immunocytochemistry/ Immunofluorescence
Sample Fruit fly (Drosophila melanogaster) Cell (Drosophila embryo)
Permeabilization No
Specification Drosophila embryo
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20% · Temperature: 25°C
Fixative Formaldehyde
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Submitted Jun 17 2016

Yes, this antibody will react with RFP.

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Escherichia coli Cell lysate - other (Cells diluted to OD600 0.4)
Gel Running Conditions Reduced Denaturing (4-20%)
Loading amount 1 µg
Treatment Solulyse solution
Specification Cells diluted to OD600 0.4
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C
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Submitted Feb 29 2016

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