HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: p63 Polyclonal Antibody (STJ94912)

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

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.

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

Secondaryantibody

dilution ratioTarget band KD Visualization

time

1P0 human

corneal limbal stem cells (P0)

20µg (total protein)

STJ94912 p63 antibody

1:500

1:0000 ~77KD 60s

2P1 human

corneal limbal stem cells (P1)

20µg (total protein) ~77KD 60s1:0000

What were the results?

Figure: Lane1: P0; Lane 2: P1

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”

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