Dendra 2 anticorps

N° du produit ABIN361314

Détail du produit

Highlights:

• Dendra2 Antibody (ABIN361314) independently validated for Immunocytochemistry by Neurology Lab (INSERM).
• Publications cite use for Immunoblotting as well as Immunostaining.
• Dendra2 belongs to a new class of photoactivatable fluorescent proteins (PAFPs). PAFPs allow to photolable and track fusion proteins in vivo. Dendra2 Antibody (ABIN361314) can be used for control purposes in this setting.

Antigène: Dendra 2

Reactivité: Corail

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Inconjugué

Application: Western Blotting (WB), Flow Cytometry (FACS), Immunocytochemistry (ICC)

Specificité: The rabbit polyclonal antibody PAb (836) recognizes Dendra2 tag, a green-to red photoswitchable protein derived from an octocoral (Dendronephthya).

Purification: Purified by protein-A affinity chromatography

Pureté: > 95 % SDS

Immunogène: Dendra2 tag

Isotype: IgG

Information d'application

Indications d'application: DENDRA 2 antibody has been tested for use in Western Blotting, Flow Cytometry and Immunocytochemistry. It is expected that the antibody will be suited for all applications where recognition of the native protein is required. A titration should be performed to determine the optimal concentration

Restrictions: For Research Use only

Independent Validation (ICC)

Validation #101498 (Immunocytochemistry)

by: Plasticity in cortical networks and epilepsy, Institut du Fer à Moulin, UMR839 INSERM/UPMC

No.: #101498

Date: 03.11.2017

Antigène: Dendra2

Numéro du lot: R-053-AI-03

Application validée: Immunocytochemistry

Contrôle positif: Rat hippocampal cells transfected with a Dendra2-tagged protein (Kv2.1_Dendra)

Contrôle négative: Non transfected cells in the same preparation

Conclusion:

ABIN361314 specifically labels Dendra+ cells in hippocampal cell cultures transfected with a construct bearing Dendra2.

Validation Images

Hippocampal primary culture co-transfected with Kv2.1_Dendra and HomerDsRed (A, red fluorescence of Homer), stained with anti-Dendra2 ABIN361314 (B, revealed with A647-coupled secondary antibody) and anti-MAP2 antibody (C, revealed with A488-coupled secondary antibody). Merged images in (D), showing the labeling of anti-Dendra2 antibody (blue) that reveals the transfected cell identified by the fluorescence of HomerDsRed (red) but not the non-transfected cells (visible by the immunolabeling of MAP2, green). Scale bar: 10µm, 63x magnification.

Protocole

Anticorps primaire: ABIN361314

Anticorps secondaire: donkey anti-rabbit A647 conjugated antibody (Jackson Immunoresearch, 711-605-152, lot 123104)

Full Protocol:

• Grow hippocampal cells in Neurobasal medium 1x (Gibco, ref. 21103-049, lot 1880280) supplemented with B27 1x (Gibco, ref. 17-504-044, lot 1733317), 2mM L-glutamine (Gibco, ref. 25030-024, lot 1881896) and antibiotics (200U/ml penicillin, 200µg/ml streptomycin, Gibco, ref. 15140-122), at 37°C and 5% CO₂ at a density of 120×10³ cells/ml on 18-mm diameter glass coverslips (Assistent) pre-coated with 50µg/ml poly-D,L-ornithine (Sigma-Aldrich, P3655, lot SLBL9424V).
• Transfect cells with 0.5µg of Kv2.1_Dendra and 0.5µg of Homer_DsRed constructs (in-house made) using Transfectin Lipid transfection reagent (BioRad, ref 170-3351, lot 4206491) following the manufacturer´s instructions.
• Fix hippocampal cell cultures on coverslips with 4% buffered formalin for 15min at RT.
• Wash slides 2x in PBS (prepared from DPBS 10x, Gibco, 14200-067, lot 1754645).
• Permeabilize cells with 0.25% Triton X-100 (Roth, 3051.3) in PBS for 4min at RT.
• Wash slides 2x in PBS.
• Block non-specific binding with PBS-Goat serum 10% (Goat serum: Gibco, 16210-064, lot 1158993) for 30min at RT.
• Incubate slides with primary rabbit anti-Dendra2 antibody (antibodies-online, ABIN361314, lot R-053-AI-03) diluted 1:100 and mouse anti-MAP2 antibody (Chemicon, MAB 378, lot LV1414361) diluted 1:200 in PBS-Goat serum 10% for 1h at RT.
• Wash slides 2x 5min in PBS-Goat serum 5%.
• Incubate with secondary donkey anti-rabbit A647 conjugated antibody (Jackson Immunoresearch, 711-605-152, lot 123104) and donkey anti-mouse A488 conjugated antibody (Jackson Immunoresearch, 715-545-150, lot 123589) diluted 1:250 in PBS-Goat serum 10% for 45min at RT.
• Wash slides 1x 5min in PBS.
• Mount coverslips with 10-20µl of Mowiol antifade reagent (Calbiochem, 475904, lot D00136768).
• Image acquisition on a Nikon Eclipse Ti microscope, 63x magnification.

Notes:

Staining pattern of Kv2.1_Dendra observed with ABIN361314 reflects the staining of the native protein Kv2.1 (obtained with its specific antibody).

Stockage

Concentration: 1mg/ml

Buffer: Phosphate buffered saline (PBS) solution with 15 mM 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: Do not freeze.

Stock: 4 °C

Stockage commentaire: Store at 2-8°C.

Référence

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Détail du antigène

Antigène: Dendra 2