HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: USP-15 Polyclonal Antibody (STJ96195)

What was used?Primary antibody: STJ96195 USP-15 Antibody

Provider: St John's Laboratory

Dilution ratio: 1:1000

Application: Western Blot

Materials for validation: His6- USP15 recombinant protein, U87 cell lysate, MRC-5 cell lysate

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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 minutes in a heat block.

Gel Electrophoresis 10% 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 at on the shaker 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: 10% 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 His6-USP15 50µg (total protein)

STJ96195USP-15 antibody

1:1000 1:5000

~115KD 4s

2U87

lysate18µg(total

protein) ~115KD 4s

3 MRC-5 lysate18µg(total

protein) ~115KD 4s

What were the results?

Figure: Lane 1: U87; Lane 2: MRC-5; Lane 3: His6-USP15

What did the customer think?

Antibody Specificity:

Antibody RPPA Rating:

Testing results were provided independently by H. Kramer at the University of Oxford. They stated “In U87 and MRC-5 cell lysates, target bands were not detected by USP15 antibody (STJ96195). Positive control of His6-USP15 (50ng) gives strong signal”

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