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  1. Link

    biotin-gfp-antibody-ab6658.pdf

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Tags & Cell Markers Fusion / Marker Proteins GFP
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Biotin Anti-GFP antibody (ab6658)

  • Datasheet
  • SDS
Reviews (6)Q&A (12)References (77)

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Abpromise

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Find out more.

Western blot - Biotin Anti-GFP antibody (ab6658)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)
  • Immunohistochemistry (Frozen sections) - Biotin Anti-GFP antibody (ab6658)
  • Immunocytochemistry/ Immunofluorescence - Biotin Anti-GFP antibody (ab6658)

Key features and details

  • Biotin Goat polyclonal to GFP
  • Suitable for: WB, IP, ICC/IF, Sandwich ELISA, IHC-P, IHC-Fr
  • Reacts with: Species independent
  • Conjugation: Biotin
  • Isotype: IgG

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Overview

  • Product name

    Biotin Anti-GFP antibody
    See all GFP primary antibodies
  • Description

    Biotin Goat polyclonal to GFP
  • Host species

    Goat
  • Conjugation

    Biotin
  • Specificity

    Antibody recognizes wild type, recombinant and enhanced forms of GFP (EGFP). No reaction was observed against Human, Mouse and Rat Serum Proteins.
  • Tested applications

    Suitable for: WB, IP, ICC/IF, Sandwich ELISA, IHC-P, IHC-Frmore details
  • Species reactivity

    Reacts with: Species independent
  • Immunogen

    Recombinant full length protein corresponding to GFP aa 1-246.
    Sequence:

    MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT GKLPVPWPTL VTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTI FFKDDGNYKTRAEVKFEGDTLV NRIELKGIDFKEDGNILGHKLEYNYN SHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK


    Database link: P42212
    Run BLAST with BLAST the sequence with ExPASy Run BLAST with BLAST the sequence with NCBI
  • General notes

    Designed to detect GFP and its variants in ELISA (sandwich or capture), immunoblotting and immunoprecipitation.

    Biotinamidocaproate N-Hydroxysuccinimide Ester (BAC) Biotin/Protein Ratio: 10-20 BAC molecules per goat IgG molecule.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

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

    Preservative: 0.01% Sodium azide
    Constituents: 0.44% Potassium phosphate, 0.88% Sodium chloride

    10 mg/mL BSA, immunoglobulin and protease free
  • 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.
  • Primary antibody notes

    Designed to detect GFP and its variants in ELISA (sandwich or capture), immunoblotting and immunoprecipitation.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

    • Tags & Cell Markers
    • Fusion / Marker Proteins
    • GFP

Associated products

  • Isotype control

    • Biotin Goat IgG - Isotype Control (ab37376)
  • Recombinant Protein

    • Recombinant E. coli GFP protein (His tag) (ab119740)
    • Recombinant A. victoria GFP protein (ab84191)
  • Related Products

    • GFP ELISA Kit (ab171581)
    • Biotin Assay Kit (Colorimetric) (ab185441)
  • sELISA pair antibody

    • Anti-GFP antibody [9F9.F9] (ab1218)

Applications

Our Abpromise guarantee covers the use of ab6658 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 1/2000 - 1/10000.
IP Use at an assay dependent concentration.
ICC/IF 1/1000 - 1/5000.
Sandwich ELISA 1/10000 - 1/50000. Can be paired for Sandwich ELISA with Mouse monoclonal [9F9.F9] to GFP (ab1218).

May be used as capture or detection antibody in a Sandwich ELISA.

IHC-P 1/250.
IHC-Fr 1/5000.

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

  • Western blot - Biotin Anti-GFP antibody (ab6658)
    Western blot - Biotin Anti-GFP antibody (ab6658)
    Biotin Anti-GFP antibody (ab6658)
    Additional bands at: 28 kDa. We are unsure as to the identity of these extra bands.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)This image is a courtesy of Hongwei Shao

    ab6658 staining GFP in human melanoma cells recovered from nude mice by Immunocytochemistry/ immunoflurescence. Cells were fixed with formaldehyde, permeabilized with 0.25% Triton X-100 RT for 10min and blocking with commercially available blocking buffer was performed for 30 minutes at RT. Samples were incubated with primary antibody (1:50) for 18 hours at 4°C. An Alexa Fluor®488-conjugated donkey polyclonal to goat IgG was used as secondary antibody at 1/100 dilution. Green color indicates GFP/Fibrolast cells, while red color indicates Ki67 positive cells, most of them are tumor cells (Abcam`s ab15580 was used for the detection).

    See Abreview

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Biotin Anti-GFP antibody (ab6658)

    Immunofluorescence Microscopy using ab6658. Tissue: Drosophila melanogaster late stage embryonic central nervous system. Fixation: 0.5% PFA. Antigen retrieval: not required. Primary antibody: Anti-GFP antibody at a 1/1,000 for 1 h at RT. Secondary antibody: AlexaFluor 488™ conjugated anti-Goat antibody at 1/300 for 45 min at RT. Panel A: shows a lateral view (ventral left). Panels B and C: shows ventral views of whole mount embryos at 63x magnification (plus 2x digital zoom). In all panels, anterior is up. Staining: tau-GFP cell bodies (large arrowhead) and axons of motorneurons (arrow) and interneurons (small arrowhead) as green fluorescent signal.

  • Immunohistochemistry (Frozen sections) - Biotin Anti-GFP antibody (ab6658)
    Immunohistochemistry (Frozen sections) - Biotin Anti-GFP antibody (ab6658)

    Immunofluorescence Microscopy using ab6658. Tissue: Sf-1:Cre mice crossed to the Z/EG reporter line. Mouse brain (coronal view, 20X magnification). Fixation: 4%PFA/PBS with o/n fixation, and subsequently transferred to a 30% sucrose solution. Antigen retrieval: frozen in OCT freezing medium (Sakura) and cryostat sectioned at 40 microns. Primary antibody: Goat anti- GFP was used at 1/500 dilution in free floating imunnohistochemistry to detect GFP. Secondary antibody: Fluorchrome conjugated Anti-goat IgG secondary antibody was used for detection at 1:10,000 for 45 min at RT.Localization: Sf-1+ neurons and their processes of the ventromedial nucleus of the hypothalamus. Staining: eGFP as green fluorescent signal and sections were counterstained with DAPI.

  • Immunocytochemistry/ Immunofluorescence - Biotin Anti-GFP antibody (ab6658)
    Immunocytochemistry/ Immunofluorescence - Biotin Anti-GFP antibody (ab6658)Image courtesy of Efrat Shema by Abreview.

    ab6658 staining GFP in rat brain cells infected with viruses containing GFP under a CMV promoter by Immunocytochemistry/ Immunofluorescence.Cells were fixed with formaldehyde, permeabilized using 0.2% Triton, blocked with 20% serum and then incubated with ab6658 at a 1:50 dilution for 20 hours at 25°C. The secondary used was an Alexa-Fluor 488 conjugated rabbit polyclonal, used at a 1/200 dilution.

    See Abreview

Protocols

  • Immunoprecipitation protocols
  • Immunohistochemistry protocols
  • Immunocytochemistry & immunofluorescence protocols
  • Western blot protocols

Click here to view the general protocols

Datasheets and documents

    • Datasheet
    • SDS
  • References (77)

    Publishing research using ab6658? Please let us know so that we can cite the reference in this datasheet.

    ab6658 has been referenced in 77 publications.

    • He S  et al. In vivo single-cell lineage tracing in zebrafish using high-resolution infrared laser-mediated gene induction microscopy. Elife 9:N/A (2020). PubMed: 31904340
    • Wasserman D  et al. Cell cycle oscillators underlying orderly proteolysis of E2F8. Mol Biol Cell 31:725-740 (2020). PubMed: 31995441
    • Chang Z  et al. Microfluidic Synchronizer Using a Synthetic Nanoparticle-Capped Bacterium. ACS Synth Biol 8:962-967 (2019). PubMed: 30964646
    • Lee JY  et al. Limiting Neuronal Nogo Receptor 1 Signaling during Experimental Autoimmune Encephalomyelitis Preserves Axonal Transport and Abrogates Inflammatory Demyelination. J Neurosci 39:5562-5580 (2019). PubMed: 31061088
    • Shokri L  et al. A Comprehensive Drosophila melanogaster Transcription Factor Interactome. Cell Rep 27:955-970.e7 (2019). PubMed: 30995488
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-10 of 18 Abreviews or Q&A

    Immunoprecipitation abreview for Anti-GFP antibody (Biotin)

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Sample
    Mouse Cell lysate - whole cell (E. coli)
    Total protein in input
    4000 µg
    Immuno-precipitation step
    Other - Dynabeads
    Specification
    E. coli
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 26 2018

    Western blot abreview for Anti-GFP antibody (Biotin)

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Fibroblast)
    Gel Running Conditions
    Reduced Denaturing (10)
    Loading amount
    30 µg
    Specification
    Fibroblast
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Hongwei Shao

    Verified customer

    Submitted Sep 06 2017

    Question

    Hello,
    Is it possible to have an estimate of the binding constant (kON - kOFF) of the following product:
    ab6658

    Read More

    Abcam community

    Verified customer

    Asked on Feb 10 2015

    Answer



    I am sorry to confirm that the binding constant is not often determined for research grade antibodies and there is no binding constant information available for this particular product.

    I am sorry we have no data to share to help answer your question on this occasion.

    Read More

    Sam Washer

    Abcam Scientific Support

    Answered on Feb 10 2015

    Question

    What is the molecular weight of this biotinylated antibody?

    Read More

    Abcam community

    Verified customer

    Asked on Aug 13 2014

    Answer


    I performed the following molecular weight calculations for this antibody:

    Assuming 15 biotin molecules per IgG molecule:

    MW IgG = 150 kDa
    MW biotin = 244.31 g/mol = 244.31 Da = 0.24431 kDa
    MW ab6658 = 150 + (15*0.24431) ˜ 154 kDa

    So the molecular weight of the biotinylated antibody should be ˜ 154 kDa.

    Read More

    Kevin Hanson

    Abcam Scientific Support

    Answered on Aug 13 2014

    Question

    What percentage is conjugated to biotin?

    Read More

    Abcam community

    Verified customer

    Asked on Nov 01 2012

    Answer

    We do not determine the percentage of molecules conjugated to biotin, but it is expected to be close to 100%. The conjugation ratio between our biotin and IgG is usually 5-6 molecules of biotin per molecule of IgG.

    Read More

    Abcam Scientific Support

    Answered on Nov 01 2012

    Question


    What is the unconjugated equivalent to Anti-GFP antibody (Biotin) (ab6658)?

    Read More

    Abcam community

    Verified customer

    Asked on Sep 28 2012

    Answer

    The unconjugated version of ab6658 is ab6673.

    https://www.abcam.com/GFP-antibody-ab6673.html

    Read More

    Abcam Scientific Support

    Answered on Sep 28 2012

    Question

    Inquiry: I am using your biotin-conjugated anti-GFP (ab6658). How does the signal from this antibody compare to one that is not biotin-conjugated and requires a biotin-conjugated secondary antibody? Is the signal equal to, less than, or greater than using an unconjugated primary with a secondary? I am using ABC reagent to amplify the signal. Thanks!

    Read More

    Abcam community

    Verified customer

    Asked on Aug 30 2012

    Answer

    Thank you very much for contacting us with your question.

    In general, using a secondary antibody will help amplify the signal from the primary antibody. With a pre-conjugated primary antibody, the signal may be weaker than using a conjugated secondary antibody. The avidin-biotin complex will also help amplify the signal, and there are other benefits to using a pre-conjugated primary (no cross-reaction between the secondary and samples, faster assay, etc), so all of these points should be weighed when considering whether to use a pre-conjugated primary or a secondary antibody step.

    Read More

    Abcam Scientific Support

    Answered on Aug 30 2012

    Question

    I have a question regarding the ab6658 primary antibody. Currently, we are using it for detection of our mouse tissue.
    I would like to start using it for the detection of rat GFP now, is it possible to use this antibody or do you recommend a better one?
    If possible, I would prefer the DAB detection above the fluorescent one.
    Thanks.
    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jul 09 2012

    Answer

    >pecies specificity is not applicable for this product. You can try this antibody for detection of GFP in any type of cells if they express GFP protein.

    Read More

    Abcam Scientific Support

    Answered on Jul 09 2012

    Immunocytochemistry/ Immunofluorescence abreview for Anti-GFP antibody (Biotin)

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Rat Cell (Brain tissue slices)
    Permeabilization
    Yes - Triton 0.2%
    Specification
    Brain tissue slices
    Blocking step
    Serum as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 20% · Temperature: 25°C
    Fixative
    Formaldehyde
    Read More

    Dr. Efrat Shema

    Verified customer

    Submitted Aug 04 2011

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-GFP antibody (Biotin)

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Rat Tissue sections (Brain)
    Specification
    Brain
    Fixative
    Paraformaldehyde
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Sodium citrate pH 6.0
    Permeabilization
    Yes - Triton 0.2%
    Blocking step
    Serum as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 20%
    Read More

    Dr. Efrat Shema

    Verified customer

    Submitted Aug 02 2011

    1-10 of 18 Abreviews or Q&A

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