What is ECL used for?
ECL is considered as the experts’ detection method of choice due to its high sensitivity, excellent signal-to-noise ratio, and wide dynamic range. Additionally, ECL is also useful in quantifying a wide variety of biological materials such as cell RNA, DNA and other analytes.
How does ECL substrate work?
The ECL Western Blotting Substrate detects picogram amounts of antigen, and with the use of photographic or other imaging methods, visualizes the presence of HRP. Blots can be repeatedly exposed to X-ray film to obtain optimal results or stripped of the immunodetection reagents and reprobed.
What is ECL Western blot?
The ECL Western Blotting Substrate is a highly sensitive, nonradioactive, enhanced luminol-based chemiluminescent substrate for easy detection of horseradish peroxidase (HRP) on immunoblots. The ECL Western Blotting Substrate offers superior signal and clear background.
Does ECL expire?
Normally, the ECL signal stays stable for hours and now it is a matter of minutes. In fact, PCNA ECL signal is visible by eye in the dark room. Previously, this signal remained stable for more than at least 30 minutes.
What is ECL system?
ECL Substrates for High-Sensitivity Western Blot Detection However, luminol emits light only weakly, so enhancers are added to the reaction to increase the signal. This enhancement of a luminol-based signal is commonly referred to as enhanced chemiluminescence (ECL).
How long can chemiluminescence last?
6–24 hr.
Under optimal western blotting conditions, a chemiluminescent signal can last for 6–24 hr. The level and duration of light generation depends on the specific substrate being used and the enzyme-to-substrate ratio in the system.
How long does ECL last?
Table 1. Thermo Scientific chemiluminescent HRP substrates.
| Pierce ECL substrate | SuperSignal West Dura substrate | |
|---|---|---|
| Detection level | Low- to midpicogram | Mid-femtogram |
| Signal duration | 0.5–2 hr | 24 hr |
| Detection methods | X-ray film, CCD imager | X-ray film, CCD imager |
| Recommended primary antibody dilution* | 1:1,000 | 1:5,000 |
Is ECL toxic?
Inhalation: May be harmful if inhaled. May cause respiratory tract irritation. Skin: May be harmful if absorbed through skin.
What is ECL reagent?
Amersham™ ECL™ Western Blotting Detection Reagents were the world′s first commercial chemiluminescent reagents for Western blotting and are still the most widely used. They are at least 10-fold more sensitive than colorimetric assays and produce excellent and reliable results every time.
How is chemiluminescence detected?
Chemiluminescence Detection This most commonly used detection method involves detection using secondary antibodies conjugated with either HRP (horseradish peroxidase) or AP (alkaline phosphatase). Horseradish Peroxidase-luminol system: Luminol is a chemiluminescent substrate of HRP.
How do you remove the ECL from the membrane?
You can freeze the membrane (after washes in TBST) but you will need to probe it again with fresh antibodies after thawing. Just rinse in either would be fine. The ECL reaction will eventually “burn out”. All the substrate will be used up, and no more photons of light will be generated.
What are the enhancers used in Bio-Rad ECL?
For example, the enhancers used in Bio-Rad Clarity Max ECL substrate maximizes detection sensitivity to enable detection to the low femtogram amounts of target. The type of enhancer used can also increase the signal duration.
What is Bio-Rad clarity ECL?
The Bio-Rad Clarity ECL substrate extends the signal duration to 24 hours, permitting reimaging without loss of signal intensity. Sensitive detection down to the femtogram level of target
What are ECL substrates?
Clarity Family of ECL Western Blotting Substrates Enhanced chemiluminescence (ECL) is a highly sensitive method for detecting proteins bound to western blotting membranes. Bio-Rad’s Clarity family of ECL substrates provides simple, high-performance solutions for all your western blotting needs.
What is the best ECL substrate for Western blotting?
Bio-Rad’s Clarity family of ECL substrates provides simple, high-performance solutions for all your western blotting needs. Clarity ECL Substrate is our most versatile chemiluminescence substrate yielding high signal, low background and a long signal duration.