Ephrin B1-B3 antibody

• Catalog Number: orb614007
• Category: Antibodies
• Description: Ephrin B1-B3 antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, IF, WB
• Reactivity: Human, Mouse, Xenopus
• Isotype: IgG
• Immunogen: Anti-Ephrin B1, B2, B3 antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to C-terminal portion of human Ephrin B1, B2, B3 conjugated to Keyhole Limpet Hemocyanin (KLH).
• Concentration: 1.05 mg/mL
• Dilution range: ELISA: 1:10,000 - 50,000, IF: 1:5000, WB: 1:1000
• Form/Appearance: Liquid (sterile filtered)
• Purity: This affinity purified antibody is directed against human Ephrin B1, B2, B3. This product was affinity purified from monospecific antiserum by immunoaffinity purification. A BLAST analysis was used to suggest cross-reactivity with the antigen based on 100% homology with Ephrin-B1 to human, mouse, rat, and Gallus gallus; 100% homology with Ephrin-B2 to mouse; 100% homology with Ephrin-B3 to human and mouse.
• Conjugation: Unconjugated
• UniProt ID: P52800
• NCBI: NP_004420.1
• Storage: Store vial at -20° C or below prior to opening. This vial contains a relatively low volume of reagent (25 µL). To minimize loss of volume dilute 1:10 by adding 225 µL of the buffer stated above directly to the vial. Recap, mix thoroughly and briefly centrifuge to collect the volume at the bottom of the vial. Use this intermediate dilution when calculating final dilutions as recommended below. Store the vial at -20°C or below after dilution. Avoid cycles of freezing and thawing.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: Rabbit Anti-Ephrin B1, B2, B3 Antibody, Ephrin-B1,
• Note: For research use only
• Application notes: Anti-Ephrin B1, B2, B3 Antibody has been tested in ELISA, Western Blot, and Immunofluorescence. Expect a band at ~35-38 kDa in western blot using appropriate lysates or tissues. Positive control used Xenopus laevis and mouse kidney in WB and Xenopus laevis in IF.
• Expiration Date: 12 months from date of receipt.

Immunofluorescence of Rabbit Anti-Ephrin B1-B3 Antibody. (A). Neural Plate of Xenopus laevis stage 17. (B). Knockdown neural plate of Xenopus laevis stage 17. Primary Antibody: Anti-Ephrin B1-3 at 1:5000 overnight at 2-8°C. Secondary Antibody: Anti-Rabbit IgG Fluorescent Secondary. Fixative: 4% Paraformaldehyde in PBS. Permeabilization: 0.05% Triton-X100 in PBS. Nuclear Counterstain: None. Magnification: 63X objective, Zeiss LSM710. Results: Ephrin B1 B2 B3 triple knockdown with a mixture of all three morpholinos abolished most of the signal which can be seen at expected plasma membrane and vesicle subcellular locations.

Western Blot of Rabbit Anti-Ephrin B1-B3 Antibody. Lane 1: 1st experiment: Uninjected heterogenous embryo [+]. Lane 2: 1st experiment: Ephrin B1, 2 MO (2 morpholino oligomers). Lane 3: 2nd experiment: Ephrin B1, 2, 3 MO (3 morpholino oligomers). Lane 4: 2nd experiment: Ephrin B3 MO (1 morpholino oligomer). Lane 5: 2nd experiment: Uninjected heterogenous embryo [+]. Primary Antibody: Anti-Ephrin B1-B3 at 1:2000 overnight at 2-8°C. Secondary Antibody: Goat anti-Rabbit IgG HRP (p/n orb347654) 1:5000 for 60 min at RT. Block: 5% skim milk/TBST/0.1% Tween. Exposure: 10 sec. Predicted MW: ~38 kDa. Observed: ~37-45 kDa. Results: Ephrin B1, 2, 3 triple knockdown with a mixture of all three morpholinos abolished significant amount of the WB signal at expected size. The comparison of lane 3 and 4 suggests not much endogenous Ephrin B3 expression exist in this stage embryos.

Western Blot of Rabbit Anti-Ephrin B1-B3 Antibody. Lane 1: Opal PreStain Molecular Weight Marker. Lane 2: EPHRIN B1 overexpressing HEK293T Lysate (10 µg) [+]. Lane 3: HEK293T Lysate (10 µg) [-]. Lane 4: Mouse Kidney Lysate (p/n orb348717) (35 µg) [+]. Primary Antibody: Anti-Ephrin B1-B3 at 1:1000 overnight at 2-8°C. Secondary Antibody: Goat anti-Rabbit IgG MX10 HRP (p/n orb347654) at 1:70000 for 30 min at RT. Block: BlockOut Buffer (p/n orb348644). Predicted MW: ~38 kDa endogenous, ~37-45 kDa due to glycosylation. Observed: ~46-52 kDa overexpressed, ~34, ~40 kDa endogenous.

Western Blot of Rabbit Anti-Ephrin B1-B3 Antibody. Lane 1: Uninjected Xenopus laevis heterogenous embryo control (20 µL). Lane 2: Morpholino Knockdown of Ephrin B1/2 heterogenous embryo (20 µL). Lane 3: Morpholino Knockdown of Ephrin B1/2 heterogenous embryo (20 µL). Lane 4: Ephrin B1-HA overexpression heterogenous embryo lysate (20 µL). Lane 5: Ephrin B2-HA overexpression heterogenous embryo lysate (20 µL). Primary Antibody: Anti-Ephrin B1-B3 at 1:2000 overnight at 2-8°C. Secondary Antibody: Goat Anti-Rabbit IgG HRP (p/n orb347654) at 1:5000 for 60 mins at RT. Block: 5% skim milk/TBST/0.1% Tween for 30 mins at RT. Predicted MW: ~38 kDa. Observed MW: ~37-45 kDa (due to glycosylation). Exposure: 10 sec. Results: Ephrin B1, 2, double knockdown with a mixture of both morpholinos oligomers abolished most of the WB signal at expected size which was double checked by over-expression.