The same can also be said for other localized proteins such as those which reside in the mitochondria. These proteins rely on efficient lysing of not only the cell membrane but also the nuclear envelope to release these proteins into the lysate. Some proteins are particularly hard to extract due to their location, for example proteins that are commonly located in the nucleus such as transcription factors or histone modifiers. It also means adjustments can be made whilst imaging to ensure you capture the signal exactly as you wish, without having to worry about losing any signal.Įnsuring there is efficient extraction of the protein of interest is one step you can take right at the start of your western blot protocol. Therefore, the requirement for quick access to an imager is removed. SignalBright ECL substrate also has the benefit of providing a bright and stable signal for over 5 hours. Secondary: Quanta BioDesign HRP-Goat anti-Rabbit IgG (H&L) (11-0201-0503) at a 1:100,000 dilution. Primary antibody: Proteintech Beta Catenin 51067-2-AP, 1:20,000. Comparison of Proteintech’s SignalBright Pro (PK10011), SignalBright Plus (PK10012), and SignalBright Max (PK10013) Enhanced Chemiluminescent Substrate. The table below summarizes each SignalBright product individually, along with important aspects of their performance.įigure 2. The three different levels of sensitivity ensure the ECL substrate is well suited to the needs of the experiment taking place. These are SignalBright Pro (PK10011), SignalBright Plus (PK10012), and SignalBright Max (PK10013). There are three different SignalBright substrates available, each with a different level of sensitivity. This allows for the visualization of proteins in small sample volumes, or which are low in abundance. Using SignalBright increases the chance of generating adequate signals by providing ultra-sensitive detection and a greater signal-to-noise ratio. Proteintech has generated SignalBright, a range of enhanced chemiluminescent substrates capable of detecting femtogram levels of protein. Using a substrate that provides a brighter signal will therefore increase the chance of visualizing these low abundance proteins. These are designed to produce higher levels of chemiluminescence signal compared to your standard substrates. A highly effective way of increasing sensitivity is by using enhanced sensitivity substrates. Low abundant proteins can still be challenging to detect with standard chemiluminescent systems due to low signal sensitivity and/or low signal-to-noise ratio. Signal generation in chemiluminescent western blotting.Ī secondary antibody with an HRP conjugate enables the detection of a target protein by oxidizing its substrate luminol with the emission of light as a by-product. Because of this, it is highly recommended to use HRP and chemiluminescence when detecting these low abundant proteins.įigure 1. However, a drawback of fluorescent detection is the system is less sensitive in comparison to chemiluminescent detection. Using antibodies conjugated to a fluorophore is also a popular alternative technique since this allows for simple and easy multiplexing. One of the most common WB detection methods is achieved by using antibodies conjugated to the enzyme horseradish peroxidase (HRP), which reacts with luminol and peroxide buffer to produce light. There are a variety of western blot detection methods to utilize, each with its pros and cons. Luckily there are multiple ways in which the western blot protocol can be optimized to detect these low abundance proteins. All these considerations, along with the fact multiple assays are often required from one condition, mean the amount of sample available for western blot is frequently limited. Sometimes the nature of the assay may result in a low total protein concentration, and in many instances the sample amount itself is limited, such as the use of rare tissue samples. Not only do some proteins have low expression, but they can also be difficult to extract due to their location. There are multiple reasons why a researcher may need to detect low abundance proteins. Fortunately, there are plenty of steps you can take to increase the likelihood of observing a low abundance protein on a western blot. Despite this, detection of some low abundance proteins has proven to be tricky. It can provide a great deal of information and consequently is a powerful tool in many research settings. Western blotting is a prominent analytical technique used to study the expression and abundance of a particular protein within a sample. Why is there a need for higher sensitivity western blots?
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