HOW DID OUR ANTIBODY PERFORM?

CUSTOMER REVIEW: Actin-beta Polyclonal Antibody (STJ91464)

WHAT WAS USED?Primary antibody: Product Catalog No: STJ91464. Actin-beta polyclonal antibody. 100µl

Provider: St John's Laboratory

Dilution ratio: 1:1000

Secondary antibody:HRP goat anti-rabbit (provided by BPI)

Reference antibody:Actin (provided by BPI)

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. 

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).

WHAT WAS THE PROTOCOL?

Gel electrophoresis information： 10% polyacrylamide gel, constant voltage 160 V, 60 min.Transfer information： 0.45 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

Secondaryantibody dilution

ratio

Target band KD

Visualization time

1 HeLa lysate 10 µl STJ91464.

Actin-beta antibody

1:10001:10000 50KD 20S

2 Jurkat lysate 10 µl 1:10000 50KD 20S

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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