Antibody Customer Review for p53 Polyclonal Antibody (STJ94890)
Western Blot Antibody Customer Review for Anti-USP15 Polyclonal Antibody (STJ96195)
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Transcript of Western Blot Antibody Customer Review for Anti-USP15 Polyclonal Antibody (STJ96195)
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
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 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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