Overview

  • Product name
    Anti-GFP antibody [LGB-1]
    See all GFP primary antibodies
  • Description
    Mouse monoclonal [LGB-1] to GFP
  • Host species
    Mouse
  • Specificity
    This antibody recognizes all forms of GFP from Aquorea victoria (i.e. GFP, EGFP, YFP and CFP). See Abreview for CFP immunoprecipitation.
  • Tested applications
    Suitable for: Flow Cyt, IP, ELISA, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Recombinant full length protein corresponding to Escherichia coli GFP.
    Database link: P42212

  • 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.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

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

  • Relevance
    Function: 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

Images

  • 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
    All lanes : Sheep anti-mouse IgG HRP conjugate at 1/5000 dilution

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

  • 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 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

  • 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 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).

References

This product has been referenced in:
  • Lyytinen OL  et al. Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6. Microb Cell Fact 18:29 (2019). Read more (PubMed: 30732607) »
  • Zhang L  et al. A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling. Transl Psychiatry 8:50 (2018). Read more (PubMed: 29479060) »
See all 20 Publications for this product

Customer reviews and Q&As

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1-6 of 6 Abreviews

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

Abcam user community

Verified customer

Submitted Mar 20 2014

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

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jul 22 2010

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

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jul 22 2010

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%

Herr Dr. Vladimir Milenkovic

Verified customer

Submitted Jun 16 2010

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

Dr. Lindsay Tulloch

Verified customer

Submitted Dec 10 2009

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

Dr. Leszek Lisowski

Verified customer

Submitted Jan 06 2009

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