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Ephrin-A3 is a ligand that binds Eph receptors. This cell surface GPI-anchored protein triggers Tyrosine Kinase receptors in cell signaling. Ephrin-A3 works on a contact-dependent basis binding EpH receptors on adjacent cells leading to bidirectional signaling pathways.
1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Ephrin-A3 Polyclonal Antibody (STJ92956)
2. WHAT WAS USED? Primary antibody: Ephrin-A3 Polyclonal Antibody (STJ92956) Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: TIG-3 Cell Line Figure Target band found at 40-45kDA
3. WHAT WAS THE PROTOCOL? Treatment of materials: Cells were lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1mM NaF, 0.1 mM sodium orthovanadate) supplemented with protease inhibitors. Proteins were reduced and denatured by heating at 100¡æ for 5 min with subsequent cooling before use. Gel electrophoresis information: 15% polyacrylamide gel, constant voltage 300 V, 45 min. Transfer information: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 300 V 60 min. Blocking: blocked with 5% milk for 1 hr Primary Ab Application: 1 hour in 5% milk. Secondary Ab Application: After washing with TBST, membrane was incubated with secondary antibody for 1h at room temperature. Visualization: Enhanced chemiluminescence reagent.
4. WHAT DID THE CUSTOMER THINK? Testing results were provided by M. Takasugi, who ¡°recommend(s) the use of sensitive chemiluminescent reagent for the detection¡±. A target band for Ephrin-A3 was detected between 40-45kDa.
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