ECL Plus Western Blotting Substrate

Boster's 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). 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. Cited in 7 publication(s).

Product Info Summary

SKU AR1196-200
Pack size 200mL
Applications To detect antigen at low-picogram to mid-femtogram level by reacting with horseradish peroxidase

List of Components

DescriptionQuantityVolume Catalog Number
Reagent A: Luminol and luminous enhancer1100mL AR1196-200-A
Reagent B: Peroxide and stabilizer1100mLAR1196-200-B

Overview

Product Name ECL Plus Western Blotting Substrate
SKU/Catalog Number AR1196-200
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 Plus Western Blotting Substrate 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 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).
Equivalent Thermofisher Pierce™ ECL Plus Western Blotting Substrate (Product No. 332132, 32124), SuperSignal™ West Pico PLUS Chemiluminescent Substrate (34577, 34578, 34579, 34580);
Bio-Rad Immu-StarTM WesternCTM Chemiluminescent Kit (Product No. 170-5070)
Cite This Product ECL Plus Western Blotting Substrate (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR1196-200)
Application To detect antigen at low-picogram to mid-femtogram level by reacting with horseradish peroxidase
*Our Boster Guarantee covers the use of this product in the above tested applications.

Notes:

Type of ECL Western Blotting SubstrateSensitivity LevelCatalog Number
Hypersensitive ECL Chemiluminescent Substratelow picogramAR1170
ECL Plus Western Blotting Substratelow-picogram to mid-femtogram levelAR1196-200

Assay Principle

Boster's 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). 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.

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 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.

Troubleshooting

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

Product Images

AR1196-200 has been cited in 7 publications:

*The publications in this section are manually curated by our staff scientists. They may differ from Bioz's machine gathered results. Both are accurate. If you find a publication citing this product but is missing from this list, please let us know we will issue you a thank-you coupon.

Yi Y,Wan S,Hou Y,Cheng J,Guo J,Wang S,Khan A,Sun N,Li H.Chlorogenic acid rescues zearalenone induced injury to mouse ovarian granulosa cells. Ecotoxicol Environ Saf.2020 May;194:110401.doi:10.1016/j.ecoenv.2020.110401.Epub 2020 Mar 3.PMID:32143102.
Species: Mouse
AR1196-200 usage in article: APP:WB, SAMPLE:GCS, DILUTION:NA

Yanhong Wang,Jing Kang,Jihua Tian et al.EGFL6 Modulates WNT Signaling to Drive Esophageal Cancer Metastasis and Proliferation,23 May 2020,PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-30021/v1]
Species: Human,Mouse
AR1196-200 usage in article: APP:WB, SAMPLE:ECS, DILUTION:NA

Guo J,Ren R,Yao X,Ye Y,Sun K,Lin J,Wang G, Guo F,Xiao J,Xu T.PKM2 suppresses osteogenesis and facilitates adipogenesis by regulating β-catenin signaling and mitochondrial fusion and fission.Aging (Albany NY).2020 Feb 25;12(4):3976-3992.doi:10. 18632/aging
Species: Mouse
AR1196-200 usage in article: APP:WB, SAMPLE:BMSCS, DILUTION:NA

Tang T,Tao X,Bao X,Chen J,Dai J,Ye J,Yan Y.Mitofusin-2 (Mfn-2) Might Have Anti-Cancer Effect through Interaction with Transcriptional Factor SP1 and Consequent Regulation on Phosphatidylinositol Transfer Protein 3 (PITPNM3) Expression.Med Sci Monit.2020 J
Species: Human,Mouse
AR1196-200 usage in article: APP:WB, SAMPLE:SMMC-7721 CELL, DILUTION:NA

Zhou H,Chen A,Lu W.Corticotropin-releasing hormone reduces basal estradiol production in zebrafish follicular cells.Mol Cell Endocrinol.2021 Feb 27:111222.doi:10.1016/j.mce.2021.111222.Epub ahead of print.PMID:33652099.
Species: Zebrafish
AR1196-200 usage in article: APP:WB, SAMPLE:FOLLICULAR CELL, DILUTION:NA

Qu K,Cha H, Ru Y,Que H,Xing M.Buxuhuayu Decoction Accelerates Angiogenesis by Activating the PI3K-Akt-eNOS Signalling Pathway in a Streptozotocin-Induced Diabetic Ulcer Rat Model.J Ethnopharmacol.2021 Feb 10:113824.doi:10.1016/j.jep.2021.113824.Epub ahead
Species: Rat
AR1196-200 usage in article: APP:WB, SAMPLE:WOUND TISSUE, DILUTION:NA

Wang Y,Liu L,Tao H,Wen L,Qin S.TRPC6 participates in the development of blood pressure variability increase in sino-aortic denervated rats. Heart Vessels. 2020 Dec;35(12):1755-1765.doi: 10.1007/s00380-020-01682-1.Epub 2020 Aug 25.PMID: 32844288.
Species: Rat

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