Amersham ECL start Western Blotting Detection Reagent
Chemiluminescent detection of medium- and high-expression proteins, with a long lasting signal that provides a longer time window for detection.
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FAQ
Amersham ECL start is recommended for confirmatory Western blotting applications of high to medium levels of proteins, e g house keeping proteins, overexpressed proteins and tagged proteins.
No – Amersham ECL start gives a chemiluminescent signal output. We recommend Amersham ECL Plex for fluorescent applications as Amersham ECL Plex has the highest sensitivity for accurate quantitation– it also enables multiplexing.
Amersham ECL start Western blotting detection reagent is designed to provide high light output during chemiluminescent detection in Western blotting. The high signal intensity can fully utilize the advantages of CCD camera based imaging equipment and makes Amersham ECL start optimal for detection with ImageQuant LAS 500 and Amersham Imager 600 from Cytiva. The output light can also be detected using X-ray film (Amersham Hyperfilm™ product range).
Working solution once prepared (by mixing Solution A and Solution B in a 1:1 ratio) can be stored up to 5 days at 4 ºC and still detect high-medium protein levels.
On receipt, all components should be stored in a refrigerator at 2ºC to 8ºC. Amersham ECL start Western blotting detection reagent is sensitive to prolonged exposure to light. Always store the individual reagents in the light-tight containers in which they are provided.
Amersham ECL start Western Blotting detection reagent has two pack sizes:
RPN3243 includes reagents for 2000 cm2 membranes: Solution A (luminol solution, 100 ml) and solution B (peroxide solution, 100 ml).
RPN3244 includes reagents for 4000 cm2 membranes: Solution A (luminol solution, 200 ml) and solution B (peroxide solution, 200 ml).
Amersham ECL start is an entry-level chemiluminescent Western blotting detection reagent , suitable for confirmatory analysis of high to medium levels of proteins. Amersham ECL start is the less sensitive product within the Amersham ECL Western blotting reagent product family.
Amersham ECL start has a dynamic range (2.1) that is complementary to what is achieved using Amersham ECL. Amersham ECL Prime and Select have a higher dynamic range of 2.4 and are better adopted for accurate quantitative analysis of a wider range of protein expressions.
Amersham ECL start provides a high light output and hence the signal intensity is brighter than Amersham ECL. Dynamic range is comparable to Amersham ECL.
We recommend using Amersham Hybond™P (PVDF) membrane or Amersham Protran (NC) membrane for excellent results.
Amersham ECL start works well with commonly used blocking agents such as Amersham ECL Prime blocking reagent, Amersham ECL blocking agent, BSA, and non-fat dry milk. The choice of blocking agent should be optimized depending on application. No one blocking works optimally for all Westerns.
The kit only comes with the Luminol Solution A and Peroxide Solution B. Additional requirements for a Western blot protocol are as follows:
Required solutions:
• Dilution and wash buffer: PBS Tween™ (PBS-T) and TBS Tween (TBS-T). A Tween 20 concentration of 0.1% is suitable for most blotting applications.
Immunodetection reagents
• Primary antibody specific to the target protein(s)
• HRP conjugated secondary antibody specific to the primary antibody.
Dilute the antibodies in PBS-T or TBS-T depending on application
Amersham ECL start Western blotting detection reagent is an entry level detection reagent and optimal antibody dilution varies between antibodies depending on affinity and quality. Primary antibody dilution ranges from 1:500 -1:3000 with a stock concentration at 1mg/ml stock and secondary antibody dilution ranges from 1:5000 -1:50,000 with a stock concentration at 1 mg/ml stock.
Recommended starting exposure time is 60 seconds on a CCD imager. The exposure time can be increased or decreased depending on the obtained signal intensity.
The chemiluminescence signal produced by Amersham ECL start is very intense and is therefore preferably detected by CCD imagers such as ImageQuant LAS 500 and Amersham Imager 600. By using CCD imaging you avoid saturation, get higher sensitivity and resolution with a broader dynamic range compared to X-ray film.
It is advised to develop the film without delay. Suitable exposure time on an X-Ray film is 30 seconds. If necessary, on the basis of signal intensity obtained, exposure time for a second sheet of film can be estimated.
Amersham ECL start is compatible both with X-ray film (like Amersham Hyperfilm product range) and CCD camera detection.
We recommend 90 seconds incubation of membrane with working solution (prepared by mixing Solution A and Solution B in a 1:1 ratio) for best results. LOD remains the same with 1 minute incubation time. However, linearity of signal intensity is marginally reduced as the lowest detectable band has reduced signal intensity.