Western blotting is a popular technique in cell and molecular biology. Western blotting is used to detect the presence of a specific protein extracted from either cells or tissue.

For many years researchers have been using darkrooms and exposing their blots to film to detect the chemiluminescent signal. With the introduction of digital imaging and Western blot imaging systems and within the last decade there have been many advancements in CCD technology providing increased speed, sensitivity and quantitative data when detecting chemiluminescence. With these advancements in technology this has enabled other applications such as fluorescence western blotting to be widely used.

Western blot work flow

The western blotting technique involves the following steps: sample preparation, gel electrophoresis, membrane transfer, blocking, primary and secondary antibody, detection and analysis. The choice of membrane is very important to ensure that you have a membrane that delivers on signal without producing a high background.

Choice of membrane

When deciding on which membrane to use you need to take into consideration the membrane type, pore size and membrane format. Polyvinylidene chloride (PVDF) has the largest protein binding capacity (170 to 200mg/cm2) compared to nitrocellulose (80-100 mg/cm2) and is therefore recommended for detecting lowly expressed proteins but you can get a higher background with this membrane.

Both PVDF and nitrocellulose membranes come in typical pore sizes of 0.1, 0.2 or 0.45mm with the 0.45mm being more commonly used. Nitrocellulose membranes are ideal for detecting low molecular weight proteins whilst PVDF is more suitable for detecting higher molecular weight proteins. When performing a fluorescent multiplexed western blot, a low fluorescence PVDF membrane should be used to reduce background levels under blue and green light.

Detection method

There are three different methods of detecting proteins on a western blot: chemiluminescence, chemifluorescence and fluorescence.

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Chemiluminescence

Chemiluminescence is a popular detection method that involves a chemical reaction occurring between an enzyme e.g. horseradish peroxidase (HRP) and a chemiluminescent molecule such as luminol resulting in a light emission. This light emission can be detected with a CCD imager.

Chemiluminesce

Chemiluminesce western blot

Primary antibody (1o) binds to a target proteins on the membrane. The membrane is then incubated with a chemiluminesce substrate e.g. HRP. An enzymatic reaction occurs producing light which is detected by a CCD camera.