Overview

  • Product nameAnti-GFP antibody [LGB-1]
    See all GFP primary antibodies
  • Description
    Mouse monoclonal [LGB-1] to GFP
  • SpecificityThis antibody recognizes all forms of GFP from Aquorea victoria (i.e. GFP, EGFP, YFP and CFP). See Abreview for CFP immunoprecipitation.
  • Tested applicationsSuitable for: Flow Cyt, IP, ELISA, ICC/IF, WBmore details
  • Immunogen

    Highly purified recombinant GFP made in Escherichia coli

  • Positive control
    • ICC/IF: GFP-transfected NIH3T3 cells

Properties

Applications

Our Abpromise guarantee covers the use of ab291 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 Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
ICC/IF Use a concentration of 0.5 µg/ml.
WB Use a concentration of 0.5 µg/ml. Detects a band of approximately 27 kDa (predicted molecular weight: 27 kDa).

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 [LGB-1] images

  • ab291 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 ab291 at 1/200 dilution overnight at +4°C followed by incubation with ab150117, Goat Anti-Mouse 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 ab291 was applied gave a stronger signal in the 488 channel, indicating that ab291 is binding to GFP and therefore eliciting signal amplification.
    ab291 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).

  • Paraformaldehyde fixed COS-7 cells expressing Myr-N15-PAK2-EGFP construct (Vilas et al.(2006) PNAS 103, 6542). Myr-N15-PAK2-EGFP fluorescence is shown in green. Indirect immunofluorescenct detection of N15-PAK-EGFP using ab291 monoclonal LGB-1 anti-GFP at 0.05 ug/ml with chicken anti-mouse secondary antibody conjugated to Alexa594 diluted 1/500 is shown in red. Myr-N15-PAK2-EGFP is localized to membrane ruffles and perinuclear vesicular structures (likely Golgi,TGN or late endosomes).
  • ab291 staining GFP in Dog MDCKII cells transfected with GFP by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.5% TX100 and blocked with 5% serum for 20 minutes. Samples were incubated with primary antibody (1/250 PBS + 0.1% TX100 + 1% goat serum) for 16 hours at 4°C. An Alexa Fluor®546-conjugated Goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody. DAPI was used to stain nuclei. ab291 was used to assess electoporation efficiency of double transfected MDCKII cells.

    See Abreview

  • ab291 at 6.7µg/mg lysate.
    HEK293 Cell lysate at 300µg.
    Transfected with CFP-fused protein XXX in pECFP vector. Immunoprecipitation step using Protein G.

    See Abreview

  • All lanes : Anti-GFP antibody [LGB-1] (ab291) at 0.5 µg/ml

    Lane 1 : 5ng GFP
    Lane 2 : 10ng GFP
    Lane 3 : 25ng GFP

    Secondary
    Sheep anti-mouse IgG HRP conjugate at 1/5000 dilution

    Predicted band size : 27 kDa
    Observed band size : 27 kDa

References for Anti-GFP antibody [LGB-1] (ab291)

This product has been referenced in:
  • Wypijewski KJ  et al. Identification of caveolar resident proteins in ventricular myocytes using a quantitative proteomic approach: dynamic changes in caveolar composition following adrenoceptor activation. Mol Cell Proteomics 14:596-608 (2015). Read more (PubMed: 25561500) »
  • Huang H  et al. Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy. PLoS One 10:e0121777 (2015). Read more (PubMed: 25811483) »

See all 10 Publications for this product

Product Wall

mutant GFP-cycle3 has not been tested with this antibody

Application Immunohistochemistry (Frozen sections)
Blocking step NOVOCASTRA protein block as blocking agent as blocking agent as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C
Sample Mouse Tissue sections (E10.5 mouse embryo)
Specification E10.5 mouse embryo
Permeabilization Yes - PBS / 0.5% v/v Triton X-100 (Sigma)
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Mar 20 2014

Ab291 should be detecting nanomolar amounts of antigen, and very likely picomolar as well in ELISA. In Western blotting it readily detects 5ng GFP (0.2 picomoles) in a film exposure of only a few seconds.

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - whole cell (HEK 293 cells transfected with Rab27a-GFP)
Total protein in input 25 µg
Specification HEK 293 cells transfected with Rab27a-GFP
Immuno-precipitation step Protein G
Username

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jul 22 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (HEK 293 cells transfected with Rab27a-GFP)
Loading amount 25 µg
Specification HEK 293 cells transfected with Rab27a-GFP
Gel Running Conditions Reduced Denaturing (10%)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jul 22 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Dog Cell (MDCK II cells transfected with GFP)
Specification MDCK II cells transfected with GFP
Fixative Paraformaldehyde
Permeabilization Yes - 0.5% TX100
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 5%
Username

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jun 16 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - other (HEK 293)
Total protein in input 300 µg
Specification HEK 293
Treatment Transfected with CFP-fused protein XXX in pECFP vector
Immuno-precipitation step Protein G
Username

Dr. Lindsay Tulloch

Verified customer

Submitted Dec 10 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Hela, Huh7 and 293 cells)
Loading amount 10 µg
Specification Hela, Huh7 and 293 cells
Gel Running Conditions Reduced Denaturing (4-12% Bis-Tris Gel)
Blocking step Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Dr. Leszek Lisowski

Verified customer

Submitted Jan 06 2009

Adjuvant was used with this ab290 and may not be suitable for your research. However, we have a few other products that you may be interested in. 1) ab291 GFP antibody [LGB-1]: No adjuvant was used 2) ab5450 GFP antibody: The affinity purificati...

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We have been unable to find any information on whether the estrogen receptor is expressed in brain - this antibody was primarily produced for the diagnosis of breast tumours. Secondly, although this antibody can be used in frozen (cryostat) sections...

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