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SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity)

(HB9308)
Technical documents: Datasheet

Product overview

Name SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity)
Biological description

Overview

Hello Bio SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) is a high performance enhanced chemiluminescent (ECL) substrate suitable for Western blotting of low abundance proteins in complex mixtures with horseradish peroxidase (HRP) conjugated secondary antibodies. 

Key Features

Sensitivity: Equivalent to ThermoFisher SuperSignal™ West Femto Maximum Sensitivity Substrate
Stability: 1 year at 4°C
Compatability: 
Ideal for film and digital imaging. Compatible with PVDF and nitrocellulose membranes and all blocking solutions, primary and secondary antibodies.

SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) produces an excellent signal/noise ratio with low background to produce publication quality Western blots. 

Notes

We recommend:

  • 100ml (50ml part A + 50ml part B) for developing around 65 blots of 10x7.5cm size (≈5,000cm2 of membrane)
  • 200ml (100ml part A + 100ml part B) for developing around 130 blots of 10x7.5cm size (≈10,000cm2 of membrane)
  • 500ml (250ml part A + 250ml part B) for developing around 330 blots of 10x7.5cm size (≈25,000cm2 of membrane)
Customer comments

Works beautifully, as always with any Hello Bio product! We tested this ECL Western Blotting substrate kit and it works marvelously. We get comparable result to our previous supplier's kit and are committed to switching to Hello Bio products for our ECL WB. Thank you Hello Bio as always for a wonderful product! Verified customer, Aston University

I used your ECL solution for a trial and it worked well on my blots, I liked it! Less background and less incubation time which was great. Verified customer, University of Bristol

Description

High sensitivity ECL solution for developing chemiluminescent Western blots

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Images

Figure 1. Representative blots for Neurofilament L of SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) and competitor solutions

Representative blots of Hello Bio ECL solutions and competitors showing SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) providing extremely high sensitivity with low background. Method: A 12% acrylamide gel was loaded with a dilution series of rat brain cytosol fraction (10µg to 5ng total protein per lane) and transferred to a PVDF membrane. This was then probed with a monoclonal rabbit anti-neurofilament L antibody (HB7266, 1:10,000 dilution, 100ng/ml) primary antibody and goat anti-rabbit HRP (1:10,000 dilution) secondary antibody. The blot was exposed with every ECL solution (500µl part A + 500µl part B) using a Licor Odyssey Fc digital imager . For more information on Western Blotting please see our Western blotting protocol

Figure 2. Fully counterbalanced comparison of Hello Bio ECL substrates with competitor products.

SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) shows extremely high sensitivity equivalent to Thermofisher SuperSignalTM West Femto Maximum Sensitivty Substrate. Method: 12% acrylamide gels were loaded with a dilution series of rat brain cytosol fraction (10µg to 5ng total protein per lane) and transferred to PVDF membranes. These were then probed with a monoclonal rabbit anti-neurofilament L antibody (HB7266, 1:10,000 dilution, 100ng/ml) primary antibody and goat anti-rabbit HRP (1:10,000 dilution) secondary antibody. All blots were exposed with every ECL solution (500µl part A + 500µl part B) in a fully randomised Latin square design to avoid any order effects. ECL exposure was carried out following the protocol detailed in "Application Notes". Imaging was carried out using a Licor Odyssey Fc digital imager and all band intensities were calculated in Licor Image Studio. For more information on Western Blotting please see our Western blotting protocol

Figure 3. Dot blot for neurofilament L in a complex protein mixture (rat brain cytosol fraction) using SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity)

SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) is able to detect Neurofilament L in as little as 1.5pg of rat brain cytosol fraction. Method: A nitrocellulose membrane was spotted with a dilution series of rat brain cytosol fraction (450ng to 1.5pg). Following blocking in 5% non-fat dry milk the membrane was probed with a monoclonal rabbit anti-neurofilament L antibody (HB7266, 1:10,000 dilution, 100ng/ml) primary antibody and goat anti-rabbit HRP (1:10,000 dilution) secondary antibody. The blot was exposed with SuperBlotTM ECL Western Blotting Substrate Kit (High sensitivity) (750µl part A + 750µl part B) using a Licor Odyssey Fc digital imager.

Biological Data

Application notes

Protocol for Chemiluminescent blot development with ECL

Digital Imaging
  1. Remove blot from the final wash solution and place on an imaging tray
  2. Mix equal quantities of part A and part B solutions being careful not to contaminate solutions by changing pipette tips
    1. For a 10x7.5cm gel we recommend 750µl of each solution
  3. Add combined solutions to the blot making sure to cover the entire area.
  4. Cover blot with a clear transparent sheet of plastic to prevent evaporation then immediately image.
    1. Be careful to not introduce any bubbles as these will show up in the final image.
Film Imaging
  1. Mix equal quantities of part A and part B solutions being careful not to contaminate solutions by changing pipette tips.
    1. For a 10x7.5cm gel we recommend 750µl of each solution
  2. Add combined solutions to a sheet of cling film large enough to fit the blot.
  3. Remove the blot from the final wash buffer, dab off any excess then place into the ECL solution. Use tweezers move the blot in order to soak it in ECL making sure to cover each side thoroughly.
  4. Dab off any excess ECL with filter paper then place into a pocket of clear plastic within a X-ray imaging cassette.
    1. Be careful to not introduce any bubbles as these will show up in the final image.
  5. Move to a dark room.
  6. Cut a piece of X-ray film to size then tape to the opposing door of the cassette. Close the cassette in one clean motion so that the film and blot are now in contact.
  7. Expose the film for an appropriate amount of time.
    1. This will vary depending on the target protein and which primary and secondary antibodies are used.
  8. Open the cassette and develop the film.

Solubility & Handling

Storage instructions

+4°C (protect from light)

Storage buffer

Contains 0.05% ProClin-300

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use.

Calculators

Molarity

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Dilution

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