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. To order the Otubain-1 Polyclonal Antibody (STJ97632), visit St John's Laboratory at the following link: http://www.stjohnslabs.com/otubain-1-antibody?filter_name=STJ97632

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 µm 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 µm 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
18µg(total
protein)
STJ97632.
Otubain-1
antibody
1:1000
1:5000 ~32.5KD 1s
2
MRC-5 cell
lysate
18µg(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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