HOW DID OUR ANTIBODY PERFORM?

ANTIBODY CUSTOMER REVIEW: TIMP-1 Polyclonal Antibody (STJ96023)

What was used?Primary antibody: STJ96023 TIMP-1 antibody

Provider: St John's Laboratory Ltd.

Dilution ratio: 1:500

Application: Western Blot

Materials for validation: Human corneal stromal fibroblasts

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 PVDF 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

1

Human corneal stromal

fibroblasts in serum+ medium

(HCSF+)

20µg (total protein)

STJ96023 TIMP-1 antibody

1:500

1:0000~24KD 60s

2

Human corneal stromal

fibroblasts in serum- medium

(HCSF-)

20µg (total protein) ~24KD 60s

1:0000

What were the results?

Figure: Lane 1: HCSF+; Lane 2: HCSF-

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 stromal fibroblasts cultured in medium with or with serum, visible bands were detected by STJ96023 TIMP-1 antibody ”

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