Anti-USP25 Antibody

• Catalog Number: orb654383
• Category: Antibodies
• Description: Anti-USP25 Antibody. Tested in ELISA, Flow Cytometry, IF, ICC, WB applications. This antibody reacts with Human, Monkey, Mouse, Rat.
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, ICC, IF, WB
• Reactivity: Human, Monkey, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived human USP25 recombinant protein (Position: M1-D1053).
• 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: 125 kDa
• UniProt ID: Q9UHP3
• 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: Ubiquitin carboxyl-terminal hydrolase 25; Deubiqui
• Note: For research use only
• Application notes: Western blot, 0.1-0.25μg/ml, Human, Mouse, Rat, Monkey Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human ELISA, 0.1-0.5μg/ml, -. 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 HL-60 cells using anti-USP25 antibody. Overlay histogram showing HL-60 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-USP25 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.

IF analysis of USP25 using anti-USP25 antibody. USP25 was detected in immunocytochemical section of A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL rabbit anti-USP25 Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Western blot analysis of USP25 using anti-USP25 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 HEK293 whole cell lysates, Lane 2: human placenta tissue lysates, Lane 3: monkey liver tissue lysates, Lane 4: monkey kidney tissue lysates, Lane 5: human PC-3 whole cell lysates, Lane 6: human Hela whole cell lysates, Lane 7: human A549 whole cell lysates, Lane 8: rat kidney tissue lysates, Lane 9: rat liver tissue lysates, Lane 10: rat lung tissue lysates, Lane 11: human PC-12 whole cell lysates, Lane 12: mouse lung tissue lysates, Lane 13: mouse RAW264.7 whole cell lysates, Lane 14: 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-USP25 antigen affinity purified polyclonal antibody at 0.25 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 USP25 at approximately 125 KD. The expected band size for USP25 is at 125 KD.