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One-Hour Western

Introduction

The western enhancer was designed to help you get your Western blot result

quick and easily. It only takes 1-1.5 hour to replace the 4-5 hours or overnight

process of the classical western blotting.

Features:

1. The rapid process: the kit was designed to take one hour (1/2 hour for

primary, 1/2 hour for secondary antibody binding and 8 min. washing to

obtain a desired Western blot data.

2. The time saved: It takes only 1-1.5 hour instead of the 4-5 hours classic

western process, with this kit, you can efficiently probe and re-probe

membrane for Western, and Dot blots with multiple antibodies or with

multiple concentration of an antibody for a desired result.

3. The money saving: One-Hour Western (OHW) is designed to increase

the affinity of antibody to target antigen by 6-10 times in comparison to

the classical Western method. And the used antibody in OHW antibody

binding solution can be kept at 4°C for 4-6 weeks.

For a desired Western Data:

1). Please make sure your membrane and anti-body are of good quality.

2). If the background is higher than desired, it can be modified by

increasing the percentage (2-5%) of Milk (or 1-5% BSA),

or decreasing the antibody concentration.

3). If a antibody signal is weak, it can be modified by prolonging the

exposure or increasing the antibody concentration, or using 0.5%

Milk (0.1%BSA) in 1x HHWE as antibody dilution.

Content Table

Catalog No.

Name Size Store

300-904-050 One-Hour Western Kit

1st Antibody Binding Enhancer 10X

2nd Antibody Binding Enhancer 10X

Rapid Wash buffer, 10X

50 ml/bottle x 3

At 4°C for 1 year,

Page 2 of 3

Protocol

Step Action

Step 1

• Diluted the 10X 1st Antibody Binding Enhancer (ABE) with D.W.

(H2O) to be as the 1X solution..

This solution can be kept at 5-29 °C for 6-8 weeks; at 4°C, 6 months

or longer.

• Add 2 % milk or 1% BSA to the 1X ABE to be as the ABE

1st antibody blotting solution. This solution can be kept at 4° C for

4-6 weeks.

Step 2

• Put a western blot membrane (transferred or stripped, in the ABE

antibody blotting solution with Primary antibody*, then shake at RT

for 30 min.

• Rinse the membrane with PBS-T** for 20 seconds (shaking by 3-4

times) ** PBS-T= 0.1% tween 20 in 1X PBS.

• Wash with 1X Rapid Wash buffer for 2 min.

• Wash with PBS-T for 20 seconds.

• Place the membrane in 1X 2nd antibody binding enhancer with

Secondary antibody, then shake at RT for 30 min.

• Wash with the PBS-T for 20 seconds, Repeat one more time.

• Wash with 1X Rapid Wash buffer for 5 min.

• Wash with PBS-T for 3 min. Repeat this one more time.

• Develop with substrate or by ECL, or other accepted methods.

Notes • Not guarantee for a dried used western membrane to have the

desired result.

Working Experience

Fig. 1, Anti- Fig.2-A,

 

Anti-

 

 

 

 

Fig.2-B, Anti-

 

 

 

 

 

 

Fig.3, Anti-NFkB

Actin
-Catenin(1ug/ml)
-Catenin(0.1ug/ml)

Fig.1, Mouse tissue protein, used the actin 1: 30,000.

Fig.2, Fig.2-A, Mouse tissue protein, the antibody –
-Catenin,

1ug/ml, 2 % Milk-HHWE, the result shows the high concentration

antibody used.

Page 3 of 3

Fig.2-B, The good result was obtained from the same membrane-Fig.2 A,

re-probed with Anti-B-catenin (0.1ug/ml) in 2% milk-HHWE

Fig.3 Human cell line protein, antibody-NF B p50, E-10 (Santa Cruz,

Cat#, sc-8414), 0.66ug/ml in 2% Milk-HHWE.

Reference:

1. Tibes R, et al.: Reverse phase protein array: validation of a novel

proteomic technology and utility for analysis of primary leukemia

specimens and hematopoietic stem cells. Mol Cancer Ther. 2006 Oct;

5(10):2512-21.

2. Golding MC, et al.: Suppression of prion protein in livestock by RNA

interference.Proc Natl Acad Sci U S A. 2006 Apr 4; 103(14):5285-90.

3. Bleuming SA, et al.: Altered bone morphogenetic protein signaling in

the Helicobacter pyloriinfected stomach. J. Pathol, 2006 Jun; 7-9(2):

190-7.

4. Deyde V, et al,: Identification of a monoclonal antibody from

Peromyscus maniculatus (deer mouse) cytomegalovirus (PCMV) which

binds to a protein with homology to the human CMV matrix protein

HCMV pp71. J Virol Methods. 2005 Jan;123(1):9-15.

5. Hardwick JC, et al.: Bone morphogenetic protein 2 is expressed by, and

acts upon, mature epithelial cells in the colon. Gastroenterology. 2004

Jan;126(1):111-21.

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Last modified: 09/28/09