Anti-ITCH/AIP4 Antibody

• Catalog Number: orb308791
• Category: Antibodies
• Description: Anti-ITCH/AIP4 Antibody. Tested in WB, Flow Cytometry applications. This antibody reacts with Human, Monkey, Mouse, Rat.
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: FC, WB
• Reactivity: Human, Monkey, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: A synthetic peptide corresponding to a sequence in the middle region of human ITCH/AIP4, different from the related mouse sequence by one amino acid.
• Antibody Type: Primary Antibody
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 103 kDa
• UniProt ID: Q96J02
• 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: E3 ubiquitin-protein ligase Itchy homolog; Itch; 6
• Note: For research use only
• Application notes: Western blot, 0.1-0.5μg/ml, Human, Monkey, Mouse, Rat Flow Cytometry(Fixed), 1-3 μg/1x106 cells, Human. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of SiHa cells using anti-ITCH 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 rabbit anti-ITCH Antibody (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10^6) 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 ITCH/AIP4 using anti-ITCH/AIP4 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 30 ug of sample under reducing conditions. Lane 1: human K562 whole cell lysates, Lane 2: human Raji whole cell lysates, Lane 3: human 293T whole cell lysates, Lane 4: monkey COS-7 whole cell lysates, Lane 5: human HepG2 whole cell lysates, Lane 6: human A549 whole cell lysates, Lane 7: rat C6 whole cell lysates, Lane 8: mouse NIH/3T3 whole cell 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 rabbit anti-ITCH/AIP4 antigen affinity purified polyclonal 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-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 with Tanon 5200 system. A specific band was detected for ITCH/AIP4 at approximately 103 kDa. The expected band size for ITCH/AIP4 is at 103 kDa.