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ECL reagents, known as chemiluminescent substrates for horseradish peroxidase (HRP), are currently the most sensitive reagents used in Western blotting detection. Common issues with ECL reagents on the market include quenching, high background, instability, and difficulty in detecting low-abundance proteins, which can affect the final experimental results.
Yeasen Biotech has gathered outstanding scientific researchers in the field of immunodetection. By strictly controlling the quality of luminol raw materials and continuously improving the formulation of enhancers and stabilizers, we have developed four types of ECL reagents to meet the needs of different experiments. These include enhanced, ultra-sensitive, durable, and ultra-high sensitivity products. They not only meet the detection requirements for target proteins from low to high abundance but also enhance the luminescence intensity without affecting the experiment, resulting in clearer images.
Product Features
- Superb Sensitivity — Detection of antigens from picogram to low-femtogram levels.
- Higher Signal-to-Noise Ratio — Precise luminescent substrates reduce background noise.
- More Antibody-Efficient — Optimized substrate systems for higher antibody binding affinity.
- High Cost-Performance Ratio — Higher performance at a lower price.
- Excellent Stability — New oxidants ensure stable storage at 4°C for up to one year.
Product Principle
The core principle of ECL reagent detection is the luminescence from oxidation reactions: Luminol, the main component of the luminescent substrate, is oxidized by H2O2 under alkaline conditions through the catalysis of horseradish peroxidase (HRP), forming an excited-state intermediate of 3-amino-phthalic acid. When it returns to the ground state, photons are emitted, with a maximum emission wavelength of 425 nm. These photon signals can be captured by X-ray film or CCD imaging devices.
Selection Guide
Yeasen currently offers four types of ECL products, each with its unique features, to fully meet the needs of users' immunoblotting applications.
So, which product should you choose for your experiment?
Table 1. ECL Reagent Selection Guide
|
Yeasen |
Enhanced ECL Enhanced Type |
Super ECL Ultra-Sensitive Type |
SuperDura Durable Type |
MaxiSignal Ultra-High Sensitivity Type |
|
Applicable Samples |
Target with high abundance, routine protein samples |
Target with low abundance, limited samples |
Target with low abundance, limited samples |
Target with extremely low abundance, precious samples |
|
Detection Sensitivity |
Medium picogram level |
Low picogram level |
Medium femtogram level |
Low femtogram level |
|
Signal Duration |
< 2 h |
< 12 h |
< 24 h |
< 8 h |
|
Applicable Detection System |
All reagents are suitable for X-ray film, CCD imaging devices |
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Primary Antibody Dilution Concentration |
1:1000-1:4000 |
1:1000-1:10,000 |
1:1000-1:50,000 |
1:5000-1:100,000 |
|
Secondary Antibody Dilution Concentration |
1:1000-1:4000 |
1:2000-1:10,000
|
1:50,000-1:250,000 |
1:100,000-1:500,000 |
|
Recommended Membrane |
NC membrane |
NC membrane or PVDF membrane |
NC membrane or PVDF membrane |
NC membrane or PVDF membrane |
|
Storage Stability |
Room temperature for 1 year |
4°C for 1 year, room temperature for half a year |
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Advantages |
Strongest signal among the same level of sensitivity |
Highest cost-performance ratio, suitable for most WB experiments |
Longest signal duration, extremely suitable for multiple exposures |
Excellent sensitivity, extremely suitable for trace protein detection |
Note: The recommended antibody dilution concentration is based on an antibody stock concentration of 1 mg/mL.
Product Application
Yeasen ECL reagents have better usage effects compared to other brands' products at the same level.
Figure 1. Comparison of detection effects between Yeasen ECL Ultra-Sensitive Substrate Super and brand T's equivalent substrate Pico.
Figure 2. Comparison of detection effects between Yeasen ECL Ultra-High Sensitivity Substrate MaxiSignal and other brands' equivalent substrate West Femto.
Figure 3. Comparison of detection effects between Yeasen ECL Durable Substrate SuperDura and other brands' equivalent substrate West Dura.
Published Literature
[1]Wang Z, Lu Z, Lin S, et al. Leucine-tRNA-synthase-2-expressing B cells contribute to colorectal cancer immunoevasion. Immunity. 2022;55(6):1067-1081.e8. doi:10.1016/j.immuni.2022.04.017(IF:43.474)
[2] Wu Z , Liu D , Pan D ,et al.Structure and engineering of miniatureAcidibacillus sulfuroxidansCas12f1[J].Nature Catalysis, 2023, 6(8):695-709.DOI:10.1038/s41929-023-00995-4(IF:38)
[3] Yao J, Wu D, Zhang C, et al. Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation. Nat Immunol. 2021;22(10):1268-1279. doi:10.1038/s41590-021-01023-y (IF:25.606)
[4] He X, Li J, Zhou G, et al. Gating of hippocampal rhythms and memory by synaptic plasticity in inhibitory interneurons. Neuron. 2021;109(6):1013-1028.e9. doi:10.1016/j.neuron.2021.01.014(IF:17.173)
Price Advantage
Figure 7. Price comparison of Yeasen ECL products with other brands' products at the same level
Frequently Asked Questions
|
Issues Encountered |
Cause Analysis |
Recommended Solutions |
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Film has reverse images |
Film has reverse images |
Film has reverse images |
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Film has brown or yellow bands |
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Fluorescence quenching |
Operation time is too long, membrane dries gradually |
Prevent membrane from drying out |
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Contamination of plastic wrap, developer, or fixer |
Develop good experimental habits |
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Insufficient HRP in the reaction system |
Increase HRP-labeled material |
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No bands or weak signal on film |
Low transfer efficiency |
Improve transfer efficiency, use pre-stained Marker for judgment |
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Insufficient or mismatched antigen/antibody |
Increase antigen/antibody amount or select appropriate antigen/antibody |
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X-ray film issues |
If the X-ray film is completely black (not transparent) after exposure, it indicates the film is fully exposed; use a new X-ray film |
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Developer/fixer issues |
Pre-expose a film to check; if there are issues, replace with new developer/fixer |
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Excessive HRP in the reaction system |
Dilute HRP-labeled material |
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High Background |
Primary and secondary antibody concentrations too high |
Reduce antibody concentration, extend blocking time |
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Antibodies not cleaned thoroughly |
Increase the number of washes |
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Incomplete blocking |
Optimize blocking conditions |
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Wrong blocking agent used |
Use a different blocking agent |
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Overexposure |
Reduce exposure time |
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Poor antibody specificity |
Replace with high-quality antibodies |
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Excessive HRP in the reaction system |
Dilute HRP-labeled material |
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Bands with empty spots |
High concentration of antigen and secondary antibody |
Dilute the sample and re-run the gel, or chill the mixed chromogenic solution and apply it to the membrane for immediate rapid development. |
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Irregular band shape |
Air bubbles during transfer or uneven hydration with methanol (for PVDF membranes) |
Optimize transfer conditions. |
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Film spots |
Aggregates of HRP-labeled material |
Filter HRP-labeled material with a 0.2μm membrane |
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Non-specific bands |
Excessive HRP in the reaction system |
Dilute HRP-labeled material |
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SDS causing non-specific protein binding |
Do not use SDS in the experimental procedure |
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Poor antibody specificity |
Replace with high-quality antibodies |
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Inadequate blocking |
Increase blocking time or use a different blocking agent |
Ordering Information
|
Product Name |
Product Number |
Specification |
|
Super ECL Detection Reagent |
36208ES60/76 |
100 mL/50 mL |
|
Enhanced ECL Chemiluminescent Substrate Kit |
36222ES60/76 |
100 mL/500 mL |
|
SuperSignal SuperDura Extended Duration Substrate |
36223ES10/60/70/76 |
10 mL/100 mL/200 mL/500mL |
|
SuperSignal MaxiSignal Maximum Sensitivity Substrate |
36224ES10/60/70/76 |
10 mL/100 mL/200 mL/500mL |