Assay Principle
(After years of development on chemiluminescence technology, there have been a variety of luminescence systems, but the most commonly used one in laboratories is still a technology system based on Luminol or its derivatives (isoluminol, etc.). Western luminescent detection reagent ECL immunoblotting chemiluminescence solution is a non-radioactive (horseradish peroxidase) luminescence system designed to detect femtogram-level trace proteins immobilized on a solid membrane (such as NC, PVDF, etc.). It is an experimental auxiliary reagent for the photosensitive recording of its immunoblotting by X-ray film (radiograph).)
West Femto Maximum Sensitivity Substrate Reagent
West Femto Maximum Sensitivity Substrate
Suggest 0.1-0.2ml of SuperLumia ECL working solution for 1 cm2 size of blot membrane.
Incubate blot 1-5 minutes in SuperLumia ECL Substrate working solution.
Drain excess reagent. Cover blot with clear plastic wrap.
Expose blot to X-ray film.
2. The diluted antibody should be stored at 4°C immediately after the antibody incubation, so that it can be reused later. While washing the HRP-labeled secondary antibody on the membrane, freshly prepare the luminescence working solution, and mix the two reagents at 1:1 ratio to prepare the working solution.
3. If the size of the blotting membrane is 1 cm2, it is recommended to use 0.1-0.2 ml of SuperKine™ West Femto Maximum Sensitivity Substrate working solution.
4. Incubate in the ECL working solution for 1-5 minutes.
5. Clamp the membrane with tweezers, and gently touch the lower edge of the membrane with the filter paper to remove excess luminescent liquid on the membrane. Cover the blotting membrane with a transparent plastic wrap.
6. Expose to X-ray film or take photos by chemiluminescence imager.
2. In order to obtain the best experiment results, you need to optimize all of your experiment elements, including the number of samples, antibody concentration, as well as the use of membranes and blocking reagents.
3. Mix the two substrate components at 1:1 ratio to prepare a substrate working solution. Please pay attention to change the tips during the aspiration process of A and B solution.
4. SuperKine™ West Femto Maximum Sensitivity Substrate has a long chemiluminescent duration, but it is best to perform compression or imaging within 30 minutes of color development.
5. It is recommended that every 1 cm2 membrane corresponds to 0.1-0.2 mL SuperKine™ West Femto Maximum Sensitivity Substrate working solution (femtogram level).
2. In the process of color development, occasionally have exposure to reverse white phenomenon, what is the reason? A : This phenomenon is due to the high protein abundance, the high concentration of HRP in the central part of the chromogenic substrate will be consumed instantaneously, resulting in non-luminescence. Suggestions: (1) Reduce the loading amount of protein and the concentration of primary and secondary antibody; (2) Reduce incubation time of luminescent liquid and shorten exposure time; (3) Change to ECL color developing solution with low sensitivity.
Assay Principle
(After years of development on chemiluminescence technology, there have been a variety of luminescence systems, but the most commonly used one in laboratories is still a technology system based on Luminol or its derivatives (isoluminol, etc.). Western luminescent detection reagent ECL immunoblotting chemiluminescence solution is a non-radioactive (horseradish peroxidase) luminescence system designed to detect femtogram-level trace proteins immobilized on a solid membrane (such as NC, PVDF, etc.). It is an experimental auxiliary reagent for the photosensitive recording of its immunoblotting by X-ray film (radiograph).)
Assay Principle
(After years of development on chemiluminescence technology, there have been a variety of luminescence systems, but the most commonly used one in laboratories is still a technology system based on Luminol or its derivatives (isoluminol, etc.). Western luminescent detection reagent ECL immunoblotting chemiluminescence solution is a non-radioactive (horseradish peroxidase) luminescence system designed to detect femtogram-level trace proteins immobilized on a solid membrane (such as NC, PVDF, etc.). It is an experimental auxiliary reagent for the photosensitive recording of its immunoblotting by X-ray film (radiograph).)
Experiment Results Display
(The sample is Mouse TNF-alpha protein (, 17KD), the primary antibody is TNF-alpha Polyclonal Antibody (, 1:2500), and the secondary antibody is HRP, Goat Anti-rabbit IgG (, 1:10000). The exposure time is 30s.)
Experiment Results Display
(The sample is Mouse TNF-alpha protein (, 17KD), the primary antibody is TNF-alpha Polyclonal Antibody (, 1:2500), and the secondary antibody is HRP, Goat Anti-rabbit IgG (, 1:10000). The exposure time is 30s.)
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Product Notes
The West Femto Maximum Sensitivity Substrate (Catalog #AAA31545) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. AAA Biotech's West Femto Maximum Sensitivity Substrate can be used in a range of immunoassay formats including, but not limited to, WB (Western Blot), IP (Immunoprecipitation). Highlight moment: Except the West Femto Maximum Sensitivity Substrate, there are many ways to improve the signal of immunological experiments, such as using SuperKine™ Enhanced Antibody Dilution Buffer ( ) in WB experiment, choosing DyLight, IFKine™ specialized secondary antibodies in IF experiment, or adopting high-quality animal serum, these are all pretty good options. Researchers should empirically determine the suitability of the West Femto Maximum Sensitivity Substrate for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "West Femto Maximum Sensitivity Substrate, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Frequently Asked Questions
What is West Femto Maximum Sensitivity Substrate used for?
West Femto Maximum Sensitivity Substrate is a high-performance enhanced chemiluminescence (ECL) substrate used in Western blotting. It is specifically designed to produce a light signal when it reacts with Horseradish Peroxidase (HRP) conjugated to secondary antibodies. This substrate is essential for researchers who need to visualize protein bands on a membrane, providing the necessary illumination for capture by X-ray film or digital imaging systems during protein analysis.
How sensitive is the West Femto substrate for Western blot detection?
As the name suggests, this substrate offers extreme sensitivity, capable of detecting proteins at the low femtogram level. This makes it significantly more powerful than standard or "entry-level" ECL substrates. Because of this high sensitivity, researchers can detect targets that are virtually invisible with other reagents, allowing for the identification of extremely low-abundance proteins while using much smaller amounts of precious primary and secondary antibodies.
When should I use West Femto instead of other ECL substrates?
You should choose West Femto when working with very low concentrations of target proteins or when your primary antibody has a low affinity. It is the ideal choice for samples where the protein of interest is naturally scarce, such as certain transcription factors or signaling molecules. AAA Biotech recommends this substrate for challenging blots where standard reagents fail to produce a visible signal, ensuring that even the faintest bands are captured.
Is West Femto suitable for detecting low-abundance proteins?
Absolutely. West Femto is specifically engineered for the detection of low-abundance proteins. Its formulation maximizes the light output from minimal enzymatic activity. This allows researchers to push the limits of Western blotting, detecting antigens that represent only a tiny fraction of the total protein load. By using this substrate, scientists can gain insights into subtle biological changes that would otherwise go unnoticed using less sensitive detection methods.
How long does the signal from West Femto substrate last?
The signal duration of West Femto is designed to be stable, typically allowing for multiple exposures over several hours. However, because the reaction is so intense, the peak signal is reached quickly. While the high-intensity light provides great sensitivity, very high concentrations of HRP can occasionally deplete the substrate locally (appearing as "ghost bands"). AAA Biotech suggests optimizing antibody dilutions to maintain a sustained, steady signal for the best imaging results.
Can West Femto substrate be used with HRP-conjugated antibodies?
Yes, West Femto is designed specifically for use with Horseradish Peroxidase (HRP) enzyme labels. It will not work with Alkaline Phosphatase (AP) or fluorescently labeled antibodies. When the HRP-conjugated secondary antibody binds to the target, the addition of West Femto triggers a chemical reaction that releases light. This compatibility makes it a standard tool for most laboratory Western blotting workflows that utilize HRP-based secondary detection systems.
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