WESTAR ECL SUBSTRATES FOR WESTERN BLOTTING

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WESTAR ECL

Introduction

WESTAR is our product line of ECL HRP substrates for Western blotting.
Our double enhancer proprietary technology allows for modulation of signal intensity and signal duration.
Each WESTAR substrate is at the top of its respective market segment regarding performance/price ratio.

Features

All WESTAR substrates are:

  • Compatible with all chemiluminescence imagers and X-ray film detection
  • Well-suited for different types of membranes and blocking reagents as well as a wide range of antibody dilutions
  • Optimized for attaining low background and high signal to noise ratio
 

Chemiluminescence is the Western blotting detection method of election in most laboratories, as it provides the greatest sensitivity and convenience for detection with film or digital imaging equipment.
Chemiluminescent substrates for horseradish (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the equal volumes of the components are mixed together.

In the chemiluminescent reaction, horseradish peroxidase catalyzes the oxidation of luminol into a reagent, which emits light when it decays. Since the oxidation of luminol is catalyzed by horseradish peroxidase, and the HRP is complexed with the protein of interest on the membrane, the amount and location of light that HRP catalyzes the emission of, is directly correlated with the location and amount of protein on the membrane.

However, the peroxide-catalyzed oxidation of luminol generates only a weak flash of light at 425 nm, which corresponds to visible blue light. The first generation of HRP detection substrates was based on the incorporation of a redox mediator or primary enhancer into the buffer, which was able to improve the enzyme turnover; thus increasing the equilibrium concentration of luminol radical anion, and thereby significantly improving the signal intensity. Recent publications have shown that by the addition of chemical compounds which act as secondary enhancers, the quality of the light emitted by the luminol oxidation can be furthermore increased, making it not only more intense but also steadier over time. Because of this breakthrough, the Italian company CYANAGEN S.r.l. has developed and patented a second generation of chemiluminescent substrates, called WESTAR.
All WESTAR substrates are protected by US7803573, EP1962095, US7855287, EP1950207, US2012009603 (A1), CA2742025, EP2405016, foreign equivalents and pending patents.

 

Product comparison


Figure1. Western blotting detection of MAP Kinase (ERK 1/2) on Hela cell lysate.

Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 10 μg to 0.16  μof total protein

Membrane: Amersham™ Protran® Premium 0,45 NC membrane (GE Healthcare)

Blocking: 5% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody:  Rabbit-anti  MAP Kinase (ERK 1/2) (SIGMA) 1:1000

Secondary antibody: Anti-Rabbit IgG HRP Linked F(ab′)2 (GE Healthcare) 1:5000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 180 seconds

 

Product comparison

Figure1Western blotting detection of HDAC-1 on Hela cell lysate.

Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 5 μg to 0.078 μg  of total protein

Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad)

Blocking: 2% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody:  Rabbit-anti Human HDAC-1 (abcam®) 1:2000

Secondary antibodyGoat anti-rabbit IgG HRP (2mg/mL) (abcam®)  1:20000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 180 seconds

 

Signal duration

 

 Figure 2. Comparison of signal intensities at time points up to 20 hours post substrate addition. Exposure time is 180 seconds for each time point (0-2-5-8 hours).

 

 Product comparison

Figure1Western blotting detection of MAP Kinase (ERK 1/2) on Hela cell lysate.

 
Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 10 μg to 0.16  μof total protein

Membrane: Amersham™ Protran® Premium 0,45 NC membrane (GE Healthcare)

Blocking: 5% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody:  Rabbit-anti  MAP Kinase (ERK 1/2) (SIGMA) 1:2500

Secondary antibody: Anti-Rabbit IgG HRP Linked F(ab′)2 (GE Healthcare) 1:50000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 180 seconds

 

 Product comparison

 Figure1Western blotting detection of HDAC-1 on Hela cell lysate.

Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 5 μg to 0.078 μg  of total protein

Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad)

Blocking: 2% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody:  Rabbit-anti Human HDAC-1 (abcam®) 1:5000

Secondary antibody: Goat anti-rabbit IgG HRP (2mg/mL) (abcam®)  1:75000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 180 seconds

 

Signal duration

Figure 2. Comparison of signal intensities at time points up to 20 hours post substrate addition. Exposure time is 180 seconds for each time point (0-2-5-8-11-20 hours).

 

 Product comparison

Figure1 Western blotting detection of HDAC1 on Hela cell lysate

Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 5μg to 0,078 μg of total protein

Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad)

Blocking: 2% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody: Rabbit-anti Human HDAC-1 (abcam®) 1:7500

Secondary antibody: Goat anti-Rabbit IgG HRP (2mg/mL) (abcam®) 1:100000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 180 seconds

 

  Signal Duration

Figure 2. Comparison of signal intensities at time points up to 20 hours post substrate addition. Exposure time is 180 seconds for each time point (0-2-5-8-11-20 hours).

 

Product comparison

Figure1Western blotting detection of HDAC-1 on  Hela cell lysate.

Sample: Two-fold dilution series of Hela whole cell lysate (abcam®) from 2.5 μg to 0,039 μg of total protein

Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad)

Blocking: 2% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody: Rabbit-anti Human HDAC-1 (abcam®) 1:15000

Secondary antibody: Goat anti-Rabbit IgG HRP (2mg/mL) (abcam®) 1:300000

Imaging: ImageQuant™ LAS 4000 (GE Healthcare)

Exposure time: 120 seconds 

 

 Signal Duration

 

 Figure 2.Comparison of signal intensities at time points up to 20 hours post substrate addition. Exposure time is 120 seconds for each time point (0-1-2-5-8-11 hours).

 

Product comparison

 

Figure1Western blotting detection of purified IKBα.

 

Sample: 1.5-fold dilution series of purified IKBα (abcam®) from 600 pg to 5.1 pg of protein

Membrane: Trans-Blot® Turbo™ Mini Nitrocellulose Transfer Packs (Biorad)

Blocking: 2% ECL™ Blocking Agent (GE Healthcare) in PBS-T

Primary antibody:  Rabbit-anti IKBα (abcam®) 1:1000

Secondary antibody: Goat anti-rabbit IgG HRP (2mg/ml) (abcam®) 1:500000

Imaging: ImageQuantTM LAS 4000 (GE Healthcare)

Exposure time: 10 seconds.

 

 

Sensitivity

 

Figure 2. Enhanced sensitivity of WESTAR HYPERNOVA compared to one of the current top-level substrates (SuperSignalTM West Femto - Thermo ScientificTM).

 

A) Purified IKBα detection with either WESTAR HYPERNOVA or SuperSignalTM West Femto. Triplicate blots for each substrate containing serial dilutions of purified IKBα were simultaneously imaged for 10 seconds with ImageQuantTM LAS 4000 (GE Healthcare).

B) Signal-to-noise ratio (S/N) analysis. The inset enlargement shows the enhanced sensitivity of WESTAR HYPERNOVA

(LOD = 5.1 pg) compared to SuperSignalTM West Femto (LOD = 39 pg).

 

(LOD = Limit of Detection)

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