Highlights

Frequently cited in scientific publications with 300+ citations
Validated inhouse for relevant applications
Extensive validation report for IF provided by one of your peers
Designed to detect RFP and its variants

Antigène

Reactivité

Discosoma

Hôte

Lapin

Clonalité

Polyclonal

Conjugué

Cet anticorp RFP est non-conjugé

Application

ELISA, Flow Cytometry (FACS), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP), Western Blotting (WB)

Fonction

Polyclonal RFP antibody is designed to detect RFP and its variants.

Réactivité croisée (Details)

Expect reactivity against RFP and its variants: mCherry, tdTomato, mBanana, mPlum, mOrange and mStrawberry. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Rabbit Serum and purified and partially purified Red Fluorescent Protein (Discosoma).
No reaction was observed against Human, Mouse or Rat serum proteins.

Purification

This product was prepared from monospecific antiserum by immunoaffinity chromatography using Red Fluorescent Protein (Discosoma) coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities.

Preadsorption: Pre-adsorbed

Stérilité

Sterile filtered

Immunogène

The immunogen is a Red Fluorescent Protein (RFP) fusion protein corresponding to the full length amino acid sequence (234aa) derived from the mushroom polyp coral Discosoma.
Immunogen Type: Recombinant Protein

Isotype

IgG

Indications d'application: Suggested dilutions:
ELISA: 1:28,700 - 1:48,700
IF: 1:200 - 1:2,000
FACS: 1:200 - 1:2,000
WB: 1:1,000 - 1:5,000
IHC: 1:200 - 1:2,000
IP: User Optimized
Commentaires: This antibody can be used to detect RFP by ELISA (sandwich or capture) for the direct binding of antigen. Biotin conjugated polyclonal anti-RFP used in a sandwich ELISA with unconjugated anti-RFP is well suited to titrate RFP in solution. The detection antibody conjugated to biotin is subsequently reacted with streptavidin conjugated HRP.
Fluorochrome conjugated polyclonal anti-RFP can be used to detect RFP by immunofluorescence microscopy in cell expression systems and can detect RFP containing inserts. Significant amplification of signal is achieved using fluorochrome conjugated polyclonal anti-RFP relative to the fluorescence of RFP alone.
For immunoblotting use either alkaline phosphatase or peroxidase conjugated polyclonal anti-RFP to detect RFP or RFP containing proteins on western blots.
Optimal titers for applications should be determined by the researcher.
Restrictions: For Research Use only

Validation #101103 (Immunofluorescence)

by: Centre d'Immunologie de Marseille-Luminy
No.: #101103
Date: 07.03.2017
Antigène: RFP
Numéro du lot: 34945
Application validée: Immunofluorescence
Contrôle positif: CX3CL1mcherry⁺ murine thymus
Contrôle négative: CX3CL1 WT murine thymus
Conclusion: Passed. The RFP antibody ABIN129578 specifically labels RFP⁺ cells in CX3CL1mcherry⁺ murine thymus in immunofluorescence, consistent with the expression pattern of CX3CL1mcherry.

Validation Images

CX3CL1 positive cells in CX3CL1mcherry murine thymus (A, mCherry) were stained with ABIN1043867 and an AF647 conjugated secondary antibody (B) or with the secondary antibody alone (C). CX3CL1 positive cells in CX3CL1 wt murine thymus were stained with ABIN1043867 and the AF647 conjugated antibody (D). Scale bar 100?m, 20x magnification, 1000 resolution

Protocole

Anticorps primaire: ABIN1043867
Anticorps secondaire: donkey Fab'2 anti-rabbit A647 conjugated antibody (Jackson Immunoresearch, 711-606-152, lot 128806)
Full Protocol: Harvest thymus from 7 weeks old mice in 1X Dulbecco's phosphate buffered saline (DPBS) (Gibco Life Technologies, 14200-067).
Fix mouse thymus in antigen fix (Diapath, P0014) for 2h at 4?C.
Wash tissue in 0.1M pH7.4 phosphate buffer at for 1h at 4?C.
Dehydrate tissue in 30% sucrose solution at 4?C ON.
Snap freeze tissue in Tissue Freezing Medium (ElectroMicroscopy Science, 72592-C) at -80?C.
Cut blocks into 25?m sections using a cryostat (Leica, CM3050 S).
Transfer sections to a slide.
Create a hydrophobic barrier on the slide around sections with Dako pen (Dako, S2002, lot 00081640).
Place slide in a humidified chamber and rehydrate sections in 0.1 M TrisHCl pH7.4 for 10min at RT.
Gently remove buffer by tapping slide.
Permeabilize tissue in 0.1M TrisHCl pH7.4 containing 2% Triton X-100 (Sigma, lot 015K0039) and 0.5% BSA, for 20min at RT.
Incubate sections with primary RFP antibody (Red Fluorescent Protein) (AA 234) (antibodies-online, ABIN129578, lot 34945) diluted 1:1000 0.1M TrisHCl pH7.4 containing 2% Triton X-100 (Sigma, batch 015K0039) and 0.5% BSA for 2h at RT.
Incubate a no primary antibody negative control in parallel in 0.1M TrisHCl pH7.4 containing 2% Triton X-100 (Sigma, batch 015K0039) and 0.5% BSA.
Wash slides 1x 5min in 0.1M TrisHCl pH7.4.
Incubate sections with secondary donkey Fab'2 anti-rabbit AF647 conjugated antibody (Jackson Immunoresearch, 711-606-152, lot 128806) diluted 1:300 in 0.1M TrisHCl pH7.4 containing 2% Triton X-100 (Sigma, batch 015K0039) and 0.5% BSA for 2h at RT.
Wash slides 1x 5min in 0.1M TrisHCl pH7.4.
Add approximately 10µL of Slowfade Gold antifade reagent (ThermoFisher Scientific, S36937, lot 1226836) for each section and mount cover slip.
Image acquisition on an LSM 880 (Zeiss), 20x magnification, 1000 resolution.
Notes

Format: Liquid
Concentration: 1.0 mg/mL
Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 0.01% (w/v) Sodium Azide
Agent conservateur: Sodium azide
Précaution d'utilisation: This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Conseil sur la manipulation: Avoid cycles of freezing and thawing.
Stock: 4 °C/-20 °C
Stockage commentaire: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
Date de péremption: 12 months

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Antigène
Autre désignation: RFP
Sujet: Synonyms: DsRed, rDsRed, Discosoma sp. Red Fluorescent Protein, Red fluorescent protein drFP583
Fluorescent proteins such as Discosoma Red Fluorescent Protein (DsRed) from sea anemone Discosoma sp. mushroom or green fluorescent protein (GFP) from Aequorea victoria jellyfish are widely used in research practice. Fusion RFP and GFP commonly serve as marker for gene expression and protein localization. As DsRed and GFP share only 19% identity, therefore, in general, anti-GFP antibodies do not recognize DsRed protein and vice versa. Structurally, Discosoma red fluorescent protein is similar to Aequorea green fluorescent protein in terms of its overall fold (a β-can) and chromophore-formation chemistry. However, Discosoma red fluorescent protein undergoes an additional step in the chromophore maturation and obligates tetrameric structure.