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.”
JOIN THE ANTIBODY VALIDATION PROJECT
1. Order any STJ9 catalogued product using this online form.
2. Test it in your laboratory using your preferred method.
3. Return a fully completed review form to receive rewards.
…in 3 simple steps…
@StJohnsLabs
http://www.slideshare.net/stjohnslabs