CSF2RA antibody (Center)

Catalog No. ABIN7300570

The Rabbit Polyclonal anti-CSF2RA antibody is suitable to detect CSF2RA in samples from Human, Mouse and Rat. It has been validated for WB. The antibody has been independently validated.

Quick Overview for CSF2RA antibody (Center) (ABIN7300570)

Target: CSF2RA (Colony Stimulating Factor 2 Receptor, Alpha, Low-Affinity (Granulocyte-Macrophage) (CSF2RA))

Reactivity: Human, Mouse, Rat

Host: Rabbit

Clonality: Polyclonal

Conjugate: This CSF2RA antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-CSF2RA Antibody

Binding Specificity: Center

Specificity: Recognizes endogenous levels of CD116 protein.

Characteristics: Rabbit polyclonal antibody to CD116

Purification: The antibody was purified by immunogen affinity chromatography.

Immunogen: KLH-conjugated synthetic peptide encompassing a sequence within the center region of human CD116.

Application Details

Application Notes: WB (1:500 - 1:1000)

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100104 (Western Blotting)

by: Instituto de Parasitología y Biomedicina López-Neyra

No.: #100104

Date: 12/23/2016

Antigen: CSF2RA

Method validated: Western Blotting

Positive Control: primary mouse microglia

Negative Control: siRNA knock-down of CSF2RA in primary mouse microglia

Notes: Passed. ABIN2705734 specifically recognizes Csf2ra in primary mouse microglia lysates.

Validation Images

Primary mouse microglia were transfected with Csf2ra siRNA and Csf2ra was revealed using ABIN2705734 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.

Full Methods

Primary Antibody: ABIN2705734

Secondary Antibody: 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 0.05nmol mouse CSF2RA Silencer Select siRNA (ThermoFisher Scientific, ASO1094M, ASO10991 and ASO109DF, 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 rabbit anti-CSF2RA antibody (antibodies-online, ABIN2705734, lot CP1D10A) diluted 1:500 in TBST 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.

Experimental Notes: The RND3 antibody ABIN2705734 reveals a protein of the expected molecular weight of mouse Csf2ra in lysates of primary mouse microglia. The protein band’s intensity decreases upon knockdown with two of the three utilized siRNAs.

Handling

Format: Liquid

Buffer: Liquid in 0.42 % Potassium phosphate, 0.87 % Sodium chloride, pH 7.3, 30 % glycerol, and 0.01 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Shipped at 4°C. Upon delivery aliquot and store at -20°C for one year. Avoid freeze/thaw cycles.

Expiry Date: 12 months

Target Details for CSF2RA

Target: CSF2RA (Colony Stimulating Factor 2 Receptor, Alpha, Low-Affinity (Granulocyte-Macrophage) (CSF2RA))

Alternative Name: CD116

Background: CSF2R, CSF2RY, Granulocyte-macrophage colony-stimulating factor receptor subunit alpha, GM-CSF-R-alpha, GMCSFR-alpha, GMR-alpha, CDw116, CD116

Gene ID: 1438, 12982

UniProt: P15509, Q00941

Pathways: JAK-STAT Signaling