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SKU CR0001-5
Pack size10mL (sufficient reagents for 2000 cm2 of membrane)
DescriptionBoster’s Hypersensitive Western Blot Chemiluminescent Substrate is a luminol-based chemiluminescent substrate and it can detect horseradish peroxidase (HRP) at high sensitivity levels (low picogram). 

List of Components

DescriptionQuantityVolumeCatalog Number
Reagent A: Luminol and enhanced chemiluminescent reagent15mL (20×)CR0001-5-A
Reagent B: peroxide and specially manufactured stable reagent15mL (20×)CR0001-5-B


Product Name Hypersensitive Western Blot Chemiluminescent Substrate
SKU/Catalog Number CR0001-5
Form Supplied Reagent A and B: Concentrated 20X solutions
Pack Size 10mL (sufficient reagents for 2000 cm2 of membrane)
Storage Upon receipt store at 4°C. Protect from light. It is stable at 4°C for one year.
Detection Method Chemiluminescent
Substrate Type HRP (Horseradish Peroxidase) Substrate
Description Boster’s Hypersensitive Western Blot Chemiluminescent Substrate is a luminol-based chemiluminescent substrate and it can detect horseradish peroxidase (HRP) at high sensitivity levels (low picogram).
Equivalent Thermofisher (Product No. 32106, 32109, 32209);
Bio-Rad Immu-StarTM HRP Chemiluminescent Kit (Product No. 170-5040, 170-5041, 170-5042, 170-5043, 170-5044, 170- 5045, 170-5046, 170-5047)
Cite This Product Hypersensitive Western Blot Chemiluminescent Substrate (Boster Biological Technology, Pleasanton CA, USA, Catalog # CR0001-5)
Application To detect antigen at low picogram level by reacting with horseradish peroxidase
*Our Boster Guarantee covers the use of this product in the above tested applications.

Assay Principle

The Boster Western Blotting Substrate is a luminol- based chemiluminescent substrate and it can detect horseradish peroxidase (HRP) at high sensitivity levels (low picogram). Boster 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.


• High signal‐to‐noise ratio, low background
• Rapid chemiluminescence
• Can operate under the daylight lamp
• High sensitivity: can detect femto antigen
• Long time chemiluminescence, can do exposure repeatedly, and optimize the test results, do not need to do western blot iteratively.
• Economize the antibody (primary and secondary antibody). For its high sensitivity, the dilution of the antibody is high.
Primary antibody: 1:1000-1:5000 or 0.2-1.0μg/ml
Secondary antibody: 1:20000-1:100000 or 10-50ng/ μl

Additional Materials Required

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.

Important product information

•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 crossreactivity 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.

Assay Protocol

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°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 chemiluminescent substrate working solution: add Reagent A and Reagent B, 50μι for each into 1ml distilled water and mix throughly. Volume: Completely submerge the membrane, per 10 cm2 membrane may need about 1ml working solution.
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.


ProblemPossible CauseSolution
Blot glows in the dark room
Membrane has brown or yellow bands
Signal fades quickly
Too much HRP in the sysem 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 sysem
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

Hypersensitive WB Chemiluminescent Substrate Images

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Hypersensitive WB Chemiluminescent Substrate
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Liu CL, Deng ZY, Du ER, Xu CS. Mol Med Rep. 2018 Apr;17(4):5851-5859. doi: 10.3892/mmr.2018.8601. Epub 2018 Feb 13. Long non-coding RNA BC168687 small interfering RNA reduces high glucose and high free fatty acid-induced expression of P2X7 receptors in satellite glial cells

Customer Q&As

Q: How many blots (membranes) can be used with your Western Blot Chemiluminescent substrate? Keyword: quantity, applications
A: It depends on the size of your membranes. 5ml can be diluted to 100ml chemiluminescence working reagents, you may need about 1ml working solution per 10cm2 membrane.