Overview

  • Product name
  • Description
    Goat polyclonal to GFP
  • Host species
    Goat
  • Specificity
    Anti-GFP assayed by ELISA for direct binding of antigen recognizes wild type, recombinant and enhanced forms of GFP.
  • Tested applications
    Suitable for: WB, IP, ELISA, ICC/IF, IHC-P, IHC-FrFl, IHC-Frmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Fusion protein corresponding to Aequorea victoria GFP aa 1-246.
    Database link: P42212

  • Positive control
    • IHC: E5.5 Hex-GFP transgenic mouse embryo. WB: HeLa cells. Green Fluorescent protein.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 0.42% Potassium phosphate, 0.87% Sodium chloride
  • Concentration information loading...
  • Purity
    Affinity purified
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Green Fluorescent Protein (Aequorea victoria) coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab6673 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
WB 1/400 - 1/2000.

(for immunoprecipitated GFP, see Abreview).

IP Use at an assay dependent concentration.
ELISA 1/40000.

This antibody can be used to detect GFP by ELISA (sandwich or capture) for the direct binding of antigen and recognizes wild type, recombinant and enhanced forms of GFP.

ICC/IF 1/500.
IHC-P 1/200 - 1/1000.
IHC-FrFl Use at an assay dependent concentration.
IHC-Fr 1/200 - 1/1000.

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

  • Pth4:eGFP transgenic zebrafish embryos at 1 and 2 dpf were fixed with 4% PFA and washed in PBST. They were then washed in PBDT (1% BSA, 1% DMSO, 0.1% Triton X-100 in PBS, pH 7.4), blocked in 10% normal goat serum/PBDT, and incubated overnight at 4°C with primary antibodies to HuC/D (1/100) and GFP (1/400, Abcam ab6673). Further PBST washes and blocking were followed by secondary antibodies overnight at 4°C. Hoechst 34580 was added to stain nuclei (1/2500). After further PBDT and PBS washes, embryos were mounted for confocal imaging.

    Abbreviation: e, eye; hy, hypothalamus; m, midbrain; sc, spinal cord. Scale bars: 100 μm (A-C) 50 μm (D-G).

  • In utero electroporation of Disc1 and Disc1-100P constructs into wild-type neocortex and analysis at P21.

    (Panels D-E”) Expression of the constructs was assessed.

    (Panels D-D'') 2 days after transfection in vitro.

    (Panels E-E'') at P21 in vivo.

    Immunochemistry for FLAG and GFP showed that constructs encoding either WT Disc1, the Disc1-100P variant, or GFP alone, expressed these protein species in transfected HEK-293 cells in vitro (Fig 5D–5D”) and in P21 postmitotic cortical neurons in vivo (Fig 5E–5E”)

  • Immunofluorescence for assessment of GFP+ myofibers in rat tissue.

    VML affected muscle from the 50% MG + HA+LMN group were probed for the presence of GFP. GFP+ fibers were detected in a qualitatively similar magnitude at both 2 and 8 weeks post-injury indicating viable engraftment of donor derived muscle progenitor cells. Scale bars are 1mm for whole mount images, 50 μm for regions of interest.

    A portion of the TA muscle from the defect region was embedded in a talcum-based gel, frozen in 2-methylbutane, and supercooled in liquid nitrogen. Cryosections (8 μm) were prepared and stained using standard protocols for hematoxylin & eosin.

    ab6673 used at a 1/100 dilution.

  • Mouse small intestines were washed with DPBS and fixed overnight at 4°C in Zinc formalin. Following sectioning and tissue deparaffanization, antigen retrieval was performed with 10mM Tris base (pH 9.0) buffer using a pressure cooker.

    For immunohistochemistry, sections were quenched of endogenous peroxidases by 3% H2O2, and sequentially blocked with Avidin D, biotin, and protein blocking reagents. Primary antibody incubation was conducted at 4°C overnight. Secondary biotinylated antibody was added at a dilution of 1/200, and incubated 2 hours at room temperature. Finally, sections were stained according to the ABC peroxidase protocol and counterstained with hematoxylin.

    ab6673 used at a 1/200 dilution.

    Panel D: Representative anti-eGFP immunofluorescence of macroH2A WT and DKO jejunum counterstained with DAPI (blue).

  • All lanes : Anti-GFP antibody (ab6673) at 1 µg/ml (o/n at 4degC)

    Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) lysate at 10 µg
    Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) lysate at 10 µg
    Lane 3 : CHO/K1 lysate at 10 µg
    Lane 4 : MDA-MB-231 (Human breast adenocarcinoma cell line) lysate at 10 µg
    Lane 5 : A431 (Human epidermoid carcinoma cell line) lysate at 10 µg
    Lane 6 : Jurkat (Human T cell leukemia cell line from peripheral blood) lysate at 10 µg
    Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) lysate
    Lane 8 : E-coli HCP control, 50 ng
    Lane 9 : FLAG Positive control lysate at 10 µg
    Lane 10 : Red fluorescent protein, 50 ng
    Lane 11 : Green fluorescent protein, 50 ng
    Lane 12 : Glutathinoe-S-Transferase protein, 50 ng
    Lane 13 : Maltose Binding protein, 50 ng

    Secondary
    All lanes : Peroxidase goat secondary antibody, 60 min at RT at 1/30000 dilution


    Blocking Buffer: 1% Casein-TTBS for 30 min at RT.

  • E5.5 Hex-GFP transgenic mouse embryo stained for GFP using ab6673 at 1/500 dilution. Secondary antibody is a fluorochrome conjugated anti-goat IgG secondary antibody at 1/10,000 for 45 min at RT.

    Staining: GFP as green fluorescent signal with DAPI blue counterstain.

  • All lanes : Anti-GFP antibody (ab6673) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cells
    Lane 2 : Mock transfected HeLa cell lysate

    Lysates/proteins at 35 µg per lane.

    Secondary
    All lanes : IRDye® 800 conjugated Donkey-a-Goat IgG [H&L] at 1/2500 dilution

    Additional bands at: 33 kDa. We are unsure as to the identity of these extra bands.

  • All lanes : Anti-GFP antibody (ab6673) at 1/1000 dilution

    Lane 1 : MRC5VA lung fibroblast whole cell lysate overexpressing EGFP alone
    Lanes 2-3 : MRC5VA lung fibroblast whole cell lysate overexpressing an EGFP fusion protein

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : HRP-conjugated anti-goat polyclonal at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 27,55 kDa
    why is the actual band size different from the predicted?


    Exposure time: 5 seconds

    See Abreview

  • Immunofluorescence of TGN mouse liver labeling GFP on hepatocytes with ab6673.

  • Immunohistochemistry of GFP transgenic mouse liver labeling GFP with ab6673. 

References

This product has been referenced in:
See all 213 Publications for this product

Customer reviews and Q&As

11-20 of 22 Abreviews or Q&A

Answer

It seems like the anti-rabbit secondary used to detect the other primary is cross reacting with this goat primary. It is unusual to have such a high degree of cross reactivity of a secondary to a primary generated in a non-target species, but it happens. Here are a few ways to get around this issue:

1. Incubate the rabbit primary and thenanti-rabbit secondary first with the tissue. Then follow up with ab6673 and the anti-goat secondary. This way the anti-rabbit secondary will not have the chance to bind the goat primary.

2. Use an anti-rabbit seconary that has been pre-adsorbed with other mammalian sera. In this way reactivities in the secondary that bind goat primary will be removed.

3. Pre conjugate the rabbit antibody with a fluorophore, for example using our EasyLink conjugation kits (https://www.abcam.com/easylink). In this way the anti-rabbit secondary will not be required.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (pancreas)
Specification
pancreas
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate, ph6
Permeabilization
No
Blocking step
(agent) for 10 minute(s) · Concentration: 100% · Temperature: RT°C

Dr. Miri Stolovich-Rain

Verified customer

Submitted Apr 03 2012

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Liver)
Specification
Liver
Fixative
Paraformaldehyde
Antigen retrieval step
None
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C

Abcam user community

Verified customer

Submitted May 17 2011

Abreviews
Application
Western blot
Sample
Human Cell lysate - whole cell (MRC5VA Lung Fibroblasts)
Loading amount
15 µg
Specification
MRC5VA Lung Fibroblasts
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 14 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Feb 21 2011

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (mouse retina sections)
Specification
mouse retina sections
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Mr. Kevin Jin

Verified customer

Submitted Nov 19 2010

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (E10.5 Embryo)
Specification
E10.5 Embryo
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% TritonX-100 for 5 minutes
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Oct 21 2008

Answer

Thank you for your enquiry. Under suitable conditions, this product detects GFP where it is present, but we don't have any data regarding the use you have mentioned and therefore can not guarantee you will be able to obtain good results. We suggest that you refer to the latest literature to optimize conditions for your experiment. The following articles (links provided) may provide some help. Transplantation experiments: http://jap.physiology.org/cgi/content/full/95/4/1688?ck=nck http://www.jimmunol.org/cgi/content/full/176/3/1916 GDP expression in dead cells: http://cat.inist.fr/?aModele=afficheN&cpsidt=1474672

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (embryos)
Specification
embryos
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 0.1 M socium-citrate, pH 6.0
Permeabilization
Yes - PBS + 0.3% TritonX-100
Blocking step
10% donkey serum + 1% BSA in PBS with 0.3% TX-100 as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 24 2007

Answer

Yes, you may use those antibodies for your experiment. Our IP protocol can be found at the following link in pdf format: https://www.abcam.com/assets/pdf/protocols/Immunoprecipitation%20protocol%20(IP).pdf As I mentioned on the phone, either ab290 or ab6556 could be used for the IP step. I prefer unpurified polyclonal antibodies for the IP step, however, depending on the antibody and antigen purified could be better. Both antibodies have been used successfully for IP.

Read More
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Transgenic mouse brain expressing GFP in neurons)
Specification
Transgenic mouse brain expressing GFP in neurons
Fixative
Paraformaldehyde
Antigen retrieval step
None
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Mrs. Ulla Jarl

Verified customer

Submitted Nov 24 2006

11-20 of 22 Abreviews or Q&A

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