Sema6A Antibody

• Catalog Number: orb654467
• Category: Antibodies
• Description: Sema6A Antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, FC, IHC, WB
• Reactivity: Human, Mouse, Rat
• Isotype: Rabbit IgG
• Immunogen: E.coli-derived human Sema6A recombinant protein (Position: H31-N306).
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Dilution range: Western blot, 0.1-0.25μg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human Flow Cytometry, 1-3μg/1x106 cells, Human Direct ELISA, 0.1-0.5μg/ml, Human
• Form/Appearance: Lyophilized
• Conjugation: Unconjugated
• MW: 114 kDa
• UniProt ID: Q9H2E6
• Storage: Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.
• Note: For research use only
• Application notes: Tested Species: In-house tested species with positive results. Other applications have not been tested. Optimal dilutions should be determined by end users. Add 0.2ml of distilled water will yield a concentration of 500ug/ml.
• Expiration Date: 12 months from date of receipt.

Western blot analysis of Sema6A using anti-Sema6A antibody (orb654467). 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: human HEK293 whole cell lysates, Lane 2: human HELA whole cell lysates, Lane 3: human Raji whole cell lysates, Lane 4: human U20S whole cell lysates, Lane 5: human Jurkat whole cell lysates, Lane 6: human CACO-2 whole cell lysates, Lane 7: rat brain tissue lysates, Lane 8: rat PC-12 whole cell lysates, Lane 9: mouse brain tissue lysates, Lane 10: 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-Sema6A antigen affinity purified polyclonal antibody (Catalog # orb654467) 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 (Catalog # orb90503) with Tanon 5200 system. A specific band was detected for Sema6A at approximately 114KD. The expected band size for Sema6A is at 114KD.

IHC analysis of Sema6A using anti-Sema6A antibody (orb654467). Sema6A was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-Sema6A Antibody (orb654467) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # orb90444) with DAB as the chromogen.

Flow Cytometry analysis of HEPG2 cells using anti-Sema6A antibody (orb654467). Overlay histogram showing HEPG2 cells stained with orb654467 (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Sema6A Antibody (orb654467, 1μg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (5-10μg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10^6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.