Antibody Customer Review for Otubain-1 Polyclonal Antibody (STJ97632)


Target information

Otubain-1 is encoded by the OTUB1 gene and is a member of the ovarian tumour family. It is a cysteine protease that is significantly involved in the role of protein degradation through having the ability to cleave ubiquitin at Lysine 48 of branched polyubiquitin chains.

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1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: Otubain-1 Polyclonal Antibody (STJ97632)
2. What was used? Primary antibody: STJ97632 Otubain-1 Antibody Provider: St John’s Laboratory Dilution ratio: 1:1000 Application: Western Blot Materials for validation: OTUB-1 recombinant protein,U87 cell lysate, MRC-5 cell lysate Click for product data sheet PDF
3. 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 um NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 min. 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 min (the visualization time was determined by actual result. It was ensured that the visualization time was identical for the same antigen).
4. What was the protocol? Gel electrophoresis information: 12% polyacrylamide gel, constant voltage of 185V for 60 minutes. Transfer information: 0.45 um NC membrane, 1 piece of gel (wet transfer), constant voltage 40 V 120 min. No. Antigen Loading amount Primary antibody Primary antibody dilution ratio Secondary antibody dilution ratio Target band KD Visualization time 1 U87 cell lysate 18ug(total protein) STJ97632. Otubain-1 antibody 1:1000 1:5000 ~32.5KD 1s 2 MRC-5 cell lysate 18ug(total protein) STJ97632. Otubain-1 antibody 1:1000 1:5000 ~32.5KD 1s 3 OTUB-1 recombinant protein (+Ve control) 50ng (purified protein) STJ97632. Otubain-1 antibody 1:1000 1:5000 ~32.5KD 1s
5. What were the results?Marker 1 2 3 Anti-Actin (Figure: Lane1: U87; Lane 2: MRC-5; Lane 3: OTUB-1 recombinant protein).
6. What did the customer think? Antibody Specificity: Antibody Rating: Testing results were provided independently by H. Kramer at the University of Oxford. They stated ¡°OTUB-1 recombinant protein produced a strong signal, in U87 and MRC-5 cell lysates target bands were detected at longer exposures.¡±
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