CSF2RA
Reactivity: Human
WB
Host: Rabbit
Polyclonal
unconjugated
Application Notes
WB (1:500 - 1:1000)
Restrictions
For Research Use only
Validation #100104
(Western Blotting)
by
Instituto de Parasitología y Biomedicina López-Neyra
No.
#100104
Date
12/23/2016
Antigen
CSF2RA
Lot Number
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.
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.