HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: OTUB-2 Polyclonal Antibody (STJ94840)

What was used?Primary antibody: STJ94840 OTUB-2 Antibody

Provider: St John's Laboratory

Dilution ratio: 1:1000

Application: Western Blot

Materials for validation: U87 cell lysate, MRC-5 cell lysate, OTUB-2 protein as positive control

Click for product data sheet PDF

What Was The Protocol?Treatment of Materials Cell pellets were lysed in RIPA lysis buffer with protease inhibitor cocktail ( Roche) and equal

volume of 2X loading buffer (Sigma) were heated at 95˚C for 10 min in a heat block.

Gel Electrophoresis 12% Polyacrylamide gel with a constant voltage of 185V for 60 minutes.

Transfer 0.45 µm NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 minutes.

Blocking 1X TBST with 5% skimmed milk powder was used and left overnight.

Primary Antibody Probing Primary antibody was added to the solution of 1X TBST with 5% skimmed milk powder and incubated on the shaker at room temperature for 1 hour.

Membrane Wash Membrane was washed 3 times with 1X TBST, with a 5 minute incubation on the shaker in between each wash.

Secondary Antibody Probing secondary antibody was added to the solution with a 1:5000 dilution ratio with mouse mAb for a blue ladder (dilution 1:2500) for 1 hour.

Membrane Wash Membrane was washed 3 times with 1X TBST, with a 5 minute incubation on the shaker in between each wash, then finally washed with deionized water.

Visualization ECL visualization for 5 minutes (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: 12% polyacrylamide gel, constant voltage of 185V for 60 minutes.

Transfer information: 0.45 µm NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 minutes.

No. Antigen Loading amount

Primary antibody

Primary antibody

dilution ratio

Secondaryantibody

dilution ratioTarget band KD Visualization

time

1 U87 cell lysate 18µg(total protein)

STJ94840.OTUB-2 antibody

1:1000 1:5000 ~27KD 4s

2MRC-5 cell

lysate18µg(total

protein)

STJ94840.OTUB-2 antibody

1:1000 1:5000 ~27KD 4s

3 OTUB-2 protein

50ng (purified protein)

STJ94840.OTUB-2 antibody 1:1000 1:5000 ~27KD 4s

What were the results?

Anti - Actin

Marker 1 2 3

Figure: Lane1: U87: Lane 2: MRC-5, Lane 3: OTUB-2

What did the customer think?

Antibody Specificity:

Antibody Rating:

Testing results were provided independently by H. Kramer at the University of Oxford. They stated “ In U87 and MRC-5 lysate, there were target bands detected by STJ94840.”

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