|Applications||To detect antigen at low picogram level by reacting with horseradish peroxidase|
List of Components
|Reagent A: Luminol and luminous enhancer||1||100mL||AR1170-A|
|Reagent B: Peroxide and stabilizer||1||100mL||AR1170-B|
|Product Name||ECL Western Blotting Substrate|
|Form Supplied||Reagent A and B: ready-to-use 1X solutions|
|Pack Size||200mL((sufficient reagents for 2000 cm2 of membrane)|
|Storage||Upon receipt store ECL Western Blotting Substrate at 4°C. Protect from light. It is stable at 4°C for one year.|
|Substrate Type||HRP (Horseradish Peroxidase) Substrate|
|Description||Boster’s ECL Western Blotting 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||ECL Western Blotting Substrate (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1170)|
|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.
|Type of ECL Western Blotting Substrate||Sensitivity Level||Catalog Number|
|ECL Western Blotting Substrate||low picogram||AR1170|
|ECL Plus Western Blotting Substrate||low-picogram to mid-femtogram level||AR1196|
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.
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.
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 the substrate working solution by mixing Reagent A and Reagent B at 1:1. Use 0.1mL working solution per cm2 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.
|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
Insufficient protein transfer
Low HRP or substrate activity
|Increase the amount of antigen or
Optimize transfer condition
Replace the secondary antibody or substrate
|High Background||Too much HRP in the sysem
Inadequate blocking or used inappropriate blocking buffer
Use too much antigen or antibodies
Use non- specific primary antibody
|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
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
SDS caused nonspecific binding to protein
Use non-specific primary antibody
|Dilute HRP conjugate further|
Do not use SDS
Replace the antibody
Optimize blocking conditions
ECL Western Blotting Substrate Images
Click the images to enlarge.