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 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.
There are three different methods of detecting proteins on a western blot: chemiluminescence, chemifluorescence and fluorescence.
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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.
Chemifluorescence is an alternative labelling and detection method for molecular biology and biochemistry that combines fluorescence and chemiluminescence. Whilst chemiluminescence generates light based on an enzymatic reaction, chemifluorescence attaches a fluorescent molecule to either the secondary or tertiary antibody which requires excitation via a light source.
Fluorescence involves using secondary antibodies conjugated with a fluorescent molecule (fluorophore). When the fluorophore is excited by a light source, this causes the release of photons as the excited molecule returns back to its normal state which is then detected in the form of light. The light emitted from fluorophores is consistent and directly proportional to the amount of protein on the membrane. One of the advantages of using fluorescence is the ability to detect multiple targets on the same blot at the same time enabling the detection of the normalisation/loading control and protein of interest on the same blot.
Multiplex fluorescent gels
Fluorescent multiplex between two different fluorophores. Primary antibodies (1o) bind to the target proteins on the gel. The gel is then incubated with a secondary antibodies (2o) that are conjugated with different fluorophores. The gel is then imaged using Hi-LED light sources which excite each fluorophore individually and generates fluorescence for each fluorophore which is captured by a CCD camera.
For imaging your chemiluminescent or fluorescent western blots, the G:BOX Chemi range is the ideal choice. Syngene has designed a versatile range of G:BOX Chemi systems to provide researchers with high performance imaging and hassle-free automation.
Low light chemiluminescence westerns can be easily captured using GeneSys image capture software by using its binning feature to reduce exposure times. Binning combines pixels into 2×2, 3×3, 4×4, 5×5 and 6×6 formats to produce a super pixel which collects more light, increasing sensitivity or speeding up capture times. GeneSys also lets you generate one image or a series of timed images of your westerns. You can even image colorimetric molecular weight markers and automatically overlay them on your chemiluminescent image.
Using GeneSys, you can image up to five different fluorophores at a time to see them as a multi-color image or as single images.