The document summarizes an antibody customer review of a p63 polyclonal antibody (STJ94912) used to detect p63 protein in human corneal limbal stem cells. The protocol involved lysing the cells, running a gel electrophoresis at 80V for 15 minutes then 140V for 45 minutes, transferring to a membrane at 100V for 60 minutes, blocking, probing with the primary antibody at a 1:500 dilution overnight, washing, probing with a secondary antibody at a 1:10,000 dilution for 1 hour, washing again, and visualizing with an Odyssey CLx Near-Infrared Imaging System. The results showed target bands detected at ~77kD in both P0 and P1 corneal lim

1. HOW DID OUR
ANTIBODY PERFORM?
ANTIBODY CUSTOMER REVIEW: p63 Polyclonal Antibody (STJ94912)

2. What was used?
Primary antibody: STJ94912 p63 antibody
Provider: St John's Laboratory Ltd.
Dilution ratio: 1:500
Application: Western Blot
Materials for validation: Human corneal limbal stem cells
Click for product data sheet PDF

3. What Was The Protocol?
Treatment of Materials When cells were cultured to 70%-80% plating density, lysis buffer with protease inhibitors
(Roche) and equal volume of 2X loading buffer were added for 10 minutes in a water bath at
100˚C.
Gel Electrophoresis 10% polyacrylamide gel, voltage 80V for 15 minutes, then 140 V for 45 minutes (total 60
minutes).
Transfer 0.45 µm NC membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 minutes.
Blocking Odyssey Blocking Buffer (PBS) for 1 hour.
Primary Antibody Probing Primary antibody was added to the solution of Odyssey Blocking Buffer (PBS) plus 0.01% Tween
20, 4˚C overnight.
Membrane Wash Odyssey Blocking Buffer (PBS) plus 0.01% Tween-20 for 4 times.
Secondary Antibody Probing Secondary antibody was added to the solution of Odyssey Blocking Buffer (PBS) plus 0.01%
Tween-20 and 0.02% SDS with 1:10000 dilution ratio, for 1 hour.
Membrane Wash Odyssey Blocking Buffer (PBS) plus 0.01% Tween-20 for 4 times.
Visualization Odyssey CLx Near-Infrared Imaging System.

4. What was the protocol?
10% polyacrylamide gel, voltage 80V for 15 minutes, then 140 V for 45 minutes (total 60 minutes).
Transfer information: 0.45 µm NC membrane, 1 piece of gel (wet transfer), constant voltage 100 V 60 minutes.
No. Antigen
Loading
amount
Primary
antibody
Primary
antibody
dilution ratio
Secondary
antibody
dilution ratio
Target band KD
Visualization
time
1
P0 human
corneal limbal
stem cells (P0)
20µg (total
protein)
STJ94912 p63
antibody
1:500
1:0000 ~77KD 60s
2
P1 human
corneal limbal
stem cells (P1)
20µg (total
protein)
~77KD
60s1:0000

5. What were the results?
Figure: Lane1: P0; Lane 2: P1

6. What did the customer think?
Antibody Specificity:
Antibody RPPA Rating:
Testing results were provided independently by F. Abidin at Newcastle University. They
stated “In human corneal limbal stem cells, there were target bands detected by
STJ94912 p63 antibody”

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