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Beta-actin (human gene and protein symbol ACTB/ACTB) is one of six different actin isoforms which have been identified in humans. This is one of the two nonmuscle cytoskeletal actins. Actins are highly conserved proteins that are involved in cell motility, structure and integrity. Alpha actins are a major constituent of the contractile apparatus.
1. HOW DID OUR ANTIBODY PERFORM? CUSTOMER REVIEW: Actin-beta Polyclonal Antibody (STJ91464)
2. WHAT WAS USED? Primary antibody: Product Catalog No: STJ91464. Actin-beta polyclonal antibody. 100ul Provider: St John’s Laboratory Dilution ratio: 1:1000 Secondary antibody: HRP goat anti-rabbit (provided by BPI) Reference antibody: Actin (provided by BPI)
3. WHAT WAS THE PROTOCOL? Antibody: STJ91464. Actin-beta antibody Method for validation: Western Blot Materials for validation: HeLa cell line, Jurkat cell line Treatment of materials: When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors (Sigma) and equal volume of 2X loading buffer were added for 10 min water bath at 100?C.
4. WHAT WAS THE PROTOCOL? Gel electrophoresis: 10% polyacrylamide gel, constant voltage 160 V, 60 min. Transfer: 0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min. Blocking: 1X TBST with 5% skimmed milk powder for 2 hr. Primary antibody probing: primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4?C for overnight. Membrane wash: 1X TBST wash for 3 times. Secondary antibody probing: secondary antibody was added to the solution of 1X TBST with 5% skimmed milk powder with 1:10000 dilution ratio, for 1.5 hr. Membrane wash: 1X TBST wash for 3 times. Visualization: ECL visualization for 5 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
5. WHAT WAS THE PROTOCOL? Gel electrophoresis information£º10% polyacrylamide gel, constant voltage 160 V, 60 min. Transfer information£º0.45 nm PVDF membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 min. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 HeLa lysate 10 ul STJ91464. Actin-beta antibody 1:1000 1:10000 50KD 20S 2 Jurkat lysate 10 ul 1:10000 50KD 20S
6. WHAT DID THE CUSTOMER THINK? Testing results were provided by BPI laboratory. They stated that ¡°In HeLa and Jurkat cell line, there were target bands detected by STJ91464 Actin-beta Antibody¡±.
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