Anti-14-3-3 zeta/delta/YWHAZ Antibody (monoclonal, 6G5)

• Catalog Number: orb654290
• Category: Antibodies
• Description: Anti-14-3-3 zeta/delta/YWHAZ Antibody (monoclonal, 6G5). Tested in Flow Cytometry, WB applications. This antibody reacts with Human, Monkey, Mouse, Rat.
• Clonality: Monoclonal
• Species/Host: Mouse
• Isotype: Mouse IgG2b
• Conjugation: Unconjugated
• Reactivity: Human, Monkey, Mouse, Rat
• Form/Appearance: Lyophilized
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Immunogen: A synthetic peptide corresponding to a sequence in the middle region of human 14-3-3 zeta/delta, which shares 97.8% amino acid (aa) sequence identity with both mouse and rat 14-3-3 zeta/delta.
• UniProt ID: P63104
• MW: 28 kDa
• Tested applications: FC, WB
• Application notes: Western blot, 0.1-0.5μg/ml, Human, Mouse, Monkey, Rat Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Antibody Type: Primary Antibody
• Clone Number: 6G5
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: 14-3-3 protein zeta/delta; Protein kinase C inhibi
• Note: For research use only

Flow Cytometry analysis of PC-3 cells using anti-14-3-3 zeta/delta antibody. Overlay histogram showing PC-3 cells (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 mouse anti-14-3-3 zeta/delta Antibody (1 µg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x106) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of SiHa cells using anti-14-3-3 zeta/delta antibody. Overlay histogram showing SiHa cells (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 mouse anti-14-3-3 zeta/delta Antibody (1 µg/1x106 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x106 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/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 14-3-3 zeta/delta using anti-14-3-3 zeta/delta antibody. 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 50 ug of sample under reducing conditions. Lane 1: human Hela whole cell lysates; Lane 2: human A549 whole cell lysates; Lane 3: monkey COS-7 whole cell lysates; Lane 4: human Raji whole cell lysates; Lane 5: huamn Caco-2 whole cell lysates; Lane 6: huamn Jurkat whole cell lysates; Lane 7: mouse brain tissue lysates; Lane 8: rat brain tissue lysates After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-14-3-3 zeta/delta antigen affinity purified monoclonal antibody at 0.5 µg/mL overnight at 4°C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for 14-3-3 zeta/delta at approximately 28 KD. The expected band size for 14-3-3 zeta/delta is at 28 KD.