Tumor necrosis factor receptor superfamily member 6
Application Dilute:
Flow Cytometry, Optimal dilutions should be determined by end users.
Flow Cytometry analysis of RAW264.7 cells using anti-Fas antibody (A00055). Overlay histogram showing RAW264.7 cells stained with A00055 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Fas Antibody (A00055, 1µg/1x106 cells) for 30 min at 20C. DyLight®,488 conjugated goat anti-rabbit IgG (BA1127, 5-10µg/1x106 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1µg/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.
Western blot analysis of Fas using anti-Fas antibody (A00055). Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions. Lane 1: rat thymus tissue lysates, Lane 2: mouse RAW264.7 whole cell lysates. After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-Fas antigen affinity purified polyclonal antibody (Catalog A00055) at 0.5 µg/mL overnight at 4C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog EK1002) with Tanon 5200 system. A specific band was detected for Fas at approximately 37KD. The expected band size for Fas is at 37KD.
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