EHD4 Antibody

• Catalog Number: orb1676433
• Category: Antibodies
• Description: Anti-EHD4 Antibody. Tested in ELISA, Flow Cytometry, WB applications. This antibody reacts with Human, Mouse, Rat, Monkey.
• Clonality: Polyclonal
• Species/Host: Rabbit
• Isotype: Rabbit IgG
• 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.
• Purification: Immunogen affinity purified.
• Immunogen: E.coli-derived human EHD4 recombinant protein (Position: R12-D541).
• UniProt ID: Q9H223
• MW: 70 kDa
• Tested applications: ELISA, FC, WB
• Application notes: Western blot, 0.1-0.25 μg/ml, Human, Mouse, Rat, Monkey Flow Cytometry (Fixed), 1-3 μg/1x106 cells, Human ELISA, 0.1-0.5 μg/ml, -. Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml
• Cross Reactivity: No cross-reactivity with other proteins.
• Antibody Type: Primary Antibody
• 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: BAG family molecular chaperone regulator 5; BAG-5;
• Note: For research use only
• Expiration Date: 12 months from date of receipt.

Flow Cytometry analysis of U251 cells using anti-EHD4 antibody. Overlay histogram showing U251 cells (Blue line). The cells were fixed with 4% paraformaldehyde and blocked with 10% normal goat serum. And then incubated with rabbit anti-EHD4 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.

Flow Cytometry analysis of U2OS cells using anti-EHD4 antibody. Overlay histogram showing U20S 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-EHD4 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 EHD4 using anti-EHD4 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 Hela whole cell lysates, Lane 2: human HepG2 whole cell lysates, Lane 3: monkey heart tissue lysates, Lane 4: human placenta tissue lysates, Lane 5: human CACO-2 whole cell lysates, Lane 6: human PC-3 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-EHD4 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 EHD4 at approximately 70 kDa. The expected band size for EHD4 is at 61 kDa.

Western blot analysis of EHD4 using anti-EHD4 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: rat pancreas tissue lysates, Lane 2: rat heart tissue lysates, Lane 3: rat testis tissue lysates, Lane 4: rat C6 whole cell lysates, Lane 5: mouse pancreas tissue lysates, Lane 6: mouse heart tissue lysates, Lane 7: mouse testis tissue lysates, Lane 8: mouse RAW264.7 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-EHD4 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 EHD4 at approximately 70 kDa. The expected band size for EHD4 is at 61 kDa.