HOW DID OUR ANTIBODY PERFORM?

CUSTOMER REVIEW: HIF-1α Polyclonal Antibody (STJ93498)

WHAT WAS USED?

Primary antibody: STJ93498 HIF-1α PolyclonalAntibody

Provider: St John's Laboratory

Dilution ratio: 1:500

Application: Western Blot.

Materials for validation: HeLa cell line

WHAT WAS THE PROTOCOL?Treatment of materials: When cells were cultured to 90% plating density, RIPA buffer with protease inhibitors (Roche) and equal volume of 2X loading buffer were added for 10 min water bath at 100˚C.Gel electrophoresis: 4-20% polyacrylamide gel (Bio-Rad), constant voltage 180V, 40 min.Transfer: 0.25 nm nitrocellulose membrane, by using the Trans-Blot Turbo transfer system (Bio-Rad) for 7 minutes.Blocking: 1X TBST with 5% skimmed milk powder for 1 hr at room temperature.Primary antibody probing: Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder, 4˚C 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 hr.Membrane wash: 1X TBST wash for 3 times.Visualization: Chemiluminescent signal obtained by using the SuperSignal West Chemiluminescent Substrate (Pierce) were detected using the ChemiDoc™ XRS system (BioRad) with an exposure time of 1 minutes.

WHAT WAS THE PROTOCOL?

Gel electrophoresis information： 4-20% Mini-Protean TGX Gels (Bio-Rad), constant voltage 180 V, 40 min.Transfer information： 0.25 nm nitrocellulose membrane, by using the Trans-Blot Turbo transfer system (Bio-Rad) for 7 minutes.

No. Antigen Loading amount

Primary antibody

Primary antibody dilution

ratio

Secondaryantibody

dilution ratio

Target band (KDa)

Visualization time

1 HeLa lysate 20 µg

STJ93498. HIF-1α

Polyclonal Antibody

1:500 1:10000 ~92KDa 1 min

WHAT DID THE CUSTOMER THINK?

Testing results were provided by Manuela Lanzafame, PhD from Istituto di Genetica Molecolare-CNR, Italy. They stated that “In HeLa cells the STJ93498 HIF-1α antibody detects a band of the predicted molecular weight of ~92 KDa and two additional bands.”

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