RND3 anticorps (Center)

N° du produit ABIN2496515

Cet validé indépendamment anticorps Lapin Polyclonal détecte spécifiquement RND3 dans ELISA, WB et IHC. Il présente une réactivité envers Humain et Souris.

Aperçu rapide pour RND3 anticorps (Center) (ABIN2496515)

Antigène: RND3 (rho Family GTPase 3 (RND3))

Reactivité: Humain, Souris

Hôte: Lapin

Clonalité: Polyclonal

Conjugué: Cet anticorp RND3 est non-conjugé

Application: ELISA, Western Blotting (WB), Immunohistochemistry (IHC)

Détail du produit anti-RND3 anticorps

Épitope: Center

Homologie: Human Mouse

Immunogène: This ARHE antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 142~171 amino acids from the Center region of human AEHE.

Isotype: Ig Fraction

Information d'application

Indications d'application: Optimal working dilution should be determined by the investigator.

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100103 (Western Blotting)

by: Instituto de Parasitología y Biomedicina López-Neyra

No.: #100103

Date: 23.12.2016

Antigène: RND3

Numéro du lot: SH080408I

Application validée: Western Blotting

Contrôle positif: primary mouse microglia

Contrôle négative: siRNA knock-down of RND3 in primary mouse microglia

Conclusion: Passed. ABIN2496515 specifically recognizes Rnd3 in primary mouse microglia lysates.

Validation Images

Primary mouse microglia were transfected with Rnd3 siRNA and Rnd3 was revealed using ABIN2496515 as described in the protocol section (lanes 1, 2, and 3). Lysates from cells transfected with scrambled siRNA (lane 4) and untransfected cells (lane 5) served as negative controls.

Protocole

Anticorps primaire: ABIN2496515

Anticorps secondaire: anti-rabbit HRP-conjugated antibodies (DakoCytomation, P0448, lot 00055814)

Full Protocol:

• Primary mouse microglia are grown in DMEM (Invitrogen), supplemented with 10% foetal bovine serum (Gibco), 10% horse serum and penicillin and streptomycin (Gibco), at 37°C and 5% CO2 in a 12-well dish.
• Transfect cells with cells with 0.05nmol mouse RND3 Silencer Select siRNA (ThermoFisher Scientific, ASO10960, ASO109AE and ASO109FC, lot AMO04BXD) using Lipofectamine 3000 (Invitrogen) following the manufacturer´s instructions.
• Lyse cells in 25µl per well cold lysis buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1% Nonidet-P40, 1mM EDTA, 10mM NaF, 1mM Na₃VO₄, protease inhibitors (Sigma)).
• Determine total protein content of the lysates using Bradford assay (Bio-Rad, 500-0006, lot 111832).
• Denature 5µg total protein for 5min at 95°C in 5µl Laemmli SDS sample buffer and subsequently separate them on a denaturing freshly cast 10% SDS-PAGE for 1h at 140V.
• Transfer proteins onto PVDF membrane (Pall Life Sciences, 75696G, lot TO3225) with a semi-dry Western blotting system for 50min at 60mA.
• Block the membrane with TBST (TBS, 0.1% Tween) containing 5% milk ON at 4°C.
• Incubation with primary mouse anti-RND3 antibody (antibodies-online, ABIN2496515, lot SH080408I) diluted 1:500 in TBST containing 5% milk for 1h at RT.
• Wash membrane 5x 5min with TBST.
• Incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000 in TBST containing 5% milk for 1h at RT.
• Wash membrane 5x 5min with TBST and 3x 5min with TBS.
• Reveal protein bands using ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232, lot 9547481) with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.
• Strip membranes using RestoreTM Western Blot Stripping Buffer (Thermo Scientific, 21059, lot NA 164269) and subsequently incubate with a GAPDH loading control antibody (Sigma, G9545, lot 049K4787) diluted 1:10000 and with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DakoCytomation, P0448, lot 00055814) diluted 1:2000.
• Wash and reveal the protein bands using self-made ECL Western Blotting Detection Reagent with Curix RP2 Plus medical X-ray films (AGFA Health Care, ENKMV, lot 79040074) on an AGFA Health Care Curix60-developer.

Notes: The RND3 antibody ABIN2496515 reveals a protein of the expected molecular weight of murine Rnd3 in lysates of primary mouse microglia. The protein band’s intensity decreases upon siRNA knockdown.

Stockage

Format: Liquid

Stock: 4 °C,-20 °C

Stockage commentaire: Maintain refrigerated at 2-8 °C for up to 6 months. For long term storage store at -20 °C in small aliquots to prevent freeze-thaw cycles.

Date de péremption: 6 months

Détails sur RND3

Antigène: RND3 (rho Family GTPase 3 (RND3))

Autre désignation: RND3

Poids moléculaire: 27368 Da

ID gène: 390

NCBI Accession: NP_001241667

UniProt: P61587