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.
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.
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
• 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
• 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
• 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
Fig. 1, Anti- Fig.2-A,
Fig.1, Mouse tissue protein, used the actin 1: 30,000.
Fig.2,Fig.2-A, Mouse tissue protein, the antibody –
1ug/ml, 2 % Milk-HHWE, the result shows the high concentration
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.3Human cell line protein, antibody-NF B p50, E-10 (Santa Cruz,
Cat#, sc-8414), 0.66ug/ml in 2% Milk-HHWE.
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;
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):
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
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