Antibody Customer Review for OTUB-2 Polyclonal Antibody (STJ94840)


Target information

OTUB2 is encoded by the OTUB2 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 from branched polyubiquitin chains, with Lysine 63 being a favoured point of cleavage.

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1. HOW DID OUR ANTIBODY PERFORM? ANTIBODY CUSTOMER REVIEW: OTUB-2 Polyclonal Antibody (STJ94840)
2. 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
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 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).
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 minutes. 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) STJ94840. OTUB-2 antibody 1:1000 1:5000 ~27KD 4s 2 MRC-5 cell lysate 18ug(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
5. What were the results? Anti – Actin Marker 1 2 3 Figure: Lane1: U87: Lane 2: MRC-5, Lane 3: OTUB-2
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 ¡° In U87 and MRC-5 lysate, there were target bands detected by STJ94840.¡±
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