ECL Plus Western Blotting Substrate

Reagent A and B solutions

Upon receipt store ECL Plus Western Blotting Substrate at 4 degree C. Protect from light. It is stable at 4 degree C for one year.

Chemiluminescent

HRP (Horseradish Peroxidase) Substrate

ECL Plus Western Blotting Substrate is an ultra-sensitive, luminol-based chemiluminescent substrate for the detection of horseradish peroxidase (HRP) at high sensitivity levels (low picogram to mid-femtogram). Western Blotting Substrate may be used for immunoblots, western blots, dot blots and any blotting application utilizing horseradish peroxidase (HRP)-conjugates. The substrate can be used with various blocking buffers and on nitrocellulose or PVDF membranes. Such blots will exhibit low backgrounds. Produced chemiluminescence can be visualized on CCD imaging systems or x-ray film.

1. Nitrocellulose or PVDF membranes
2. X-ray film or an imaging system
3. Rotary platform shaker for agitation of membrane during incubations
4. Wash Buffer: PBS or TBS containing 0.05-0. 1% Tween-20 (PBS: 10mM Na₃PO₄, 150mM NaCl, pH7. 2; TBS: 10mM Tris, 150mM NaCl, pH7. 4)
5. Blocking Buffer: Add some blocking medium (e. g. casein, BSA or non-fat milk) into the Wash Buffer for a final blocking medium concentration of 1-5% (w/v)
6. Primary Antibody: Choose an antibody specific to the target proteins. Prepare the antibody stock solution in Wash Buffer or Blocking Buffer
7. HRP-conjugated Secondary Antibody: Choose a HRP-conjugated Secondary Antibody that specifically binds to the primary antibody.

For optimal results, use a shaking platform during incubation steps.
Do not use sodium azide as a preservative for buffers. Sodium azide is an inhibitor of HRP.
Do not handle the membrane with bare hands. Always wear gloves or use clean forceps.
All equipment must be clean and free of foreign material. Metallic devices must have no visible signs of rust. Rust may cause speckling and high background.
Exposure to the sun or any other intense light can harm the substrate. For best results keep the substrate working solution in an amber bottle and avoid prolonged exposure to any intense light. Short-term exposure to typical laboratory lighting will not harm the working solution.
Empirical testing is essential to determine the appropriate blocking reagent for each Western blot system, as cross reactivity of the blocking reagent with antibodies can cause nonspecific signal and varying system sensitivity.
When using avidin/biotin systems, avoid using milk as a blocking reagent as milk contains variable amounts of endogenous biotin, which causes high background signal.
Use sufficient volumes of wash buffer, blocking buffer, antibody solution and substrate working solution to cover the blot and ensure that it never becomes dry. Using large blocking and wash buffer volumes minimizes nonspecific signal.
Add Tween 20 Detergent (final concentration of 0.05-0.1%) to the blocking buffer and all diluted antibody solutions to minimize nonspecific signal.
Do not use polystyrene vessels to mix and prepare the substrate working solution; this type of plastic causes the solution to become cloudy and produce a precipitate.
Use new pipette tips separately while pipetting reagent A and B to avoid cross contamination.

1. Remove blot from the transfer apparatus and wash membrane with Wash Buffer 3 times for 5 minutes each.
2. Block nonspecific sites with Blocking Buffer for 60 minutes at room temperature with shaking.
3. Remove the blocking buffer and add the primary antibody working solution. Incubate blot for 1 hour at room temperature with shaking or overnight at 2-8 degree C with shaking.
4. Suspend membrane in Wash buffer and agitate for more than 5 minutes. Replace Wash Buffer at least 4-6 times. Increasing the volume of Wash Buffer, the numbers of washes and wash duration may help minimize background signal.
5. Incubate blot with the HRP conjugated secondary antibody working solution for 1 hour at room temperature with shaking.
6. Repeat Step 3 to remove nonbound HRP conjugate.
Note: The membrane must be thoroughly washed after incubation with the HRP conjugate.
7. Prepare the substrate working solution by mixing Reagent A and Reagent B at 1:1. Use 0. 1mL working solution per cm 2 of membrane.
8. Incubate blot with working solution for 1-5 minutes at room temperature.
9. Place blot in a clear plastic wrap or transparent plastic sheet protector. Use an absorbent tissue to remove excess liquid and carefully press out any bubbles from between the blot and the membrane protector.
10. Place the protected membrane in a film cassette with the protein side facing up.
11. Place X-ray film on top of the blot membrane. Perform a exposure of 1 minute, Vary the exposure time to achieve optimal results. Light emission is most intense during the first 5-30 minutes after substrate incubation.
Note: CCD detection: Put the blot membrane in the CCD and detect the chemiluminescence image according to the manufacturers' instructions.
12. Develop and fix the film.

Problem/ Possible Cause/ Solution:

*Blot glows in the dark room. Membrane has brown or yellow bands. Signal fades quickly
Too much HRP in the system
Dilute HRP conjugate further

*Weak or no signal
Used insufficient quantities of antigen or antibodies. Insufficient protein transfer. Low HRP or substrate activity
Increase the amount of antigen or antibodies. Optimize transfer condition. Replace the secondary antibody or substrate

*High Background
Too much HRP in the system. Inadequate blocking or used inappropriate blocking buffer. Insufficient washing. Use too much antigen or antibodies. Use non-specific primary antibody. Overexposed film
Dilute HRP-conjugate further. Optimize blocking conditions. Increase duration, number and volume of washes. Decrease the amount of antigen or antibodies. Replace the antibody. Decrease exposure time

*Spots within the protein bands
Ineffective protein transfer. Unevenly hydrated membrane. Bubble between X-ray film and membrane
Optimize transfer procedure. Replace the membrane. Remove all bubbles before exposing blot to film

*Speckled background
Inadequate blocking
Optimize blocking conditions

*Nonspecific bands
Too much HRP in the system. SDS caused nonspecific binding to protein. Use non-specific primary antibody. Inadequate blocking
Dilute HRP conjugate further. Do not use SDS. Replace the antibody. Optimize blocking conditions

Type of ECL Western Blotting Substrate/ Sensitivity Level
Hypersensitive ECL Chemiluminescent Substrate/ low picogram
ECL Plus Western Blotting Substrate/ low-picogram to mid-femtogram level

Assistive Reagent

Manufactured in an ISO 13485:2003 and EN ISO 13485:2012 Certified Laboratory.