Two steps
Colorimetric detection
Chemiluminescent detection
Radioactive detection
Fluorescent detection
Secondary probing
Since the membrane has been chosen for its ability to bind protein and as both antibodies and the target are proteins, steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein. Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein - typically 3-5% Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive) in Tris-Buffered Saline (TBS), with a minute percentage of detergent such as Tween 20 or Triton X-100. The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached. Thus, when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces "noise" in the final product of the Western blot, leading to clearer results, and eliminates false positives.
During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetric reaction and produces a colour. For a variety of reasons, this traditionally takes place in a two-step process, although there are now one-step detection methods available for certain applications.
Primary antibody
Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest (or a part thereof). Normally, this is part of the immune response, whereas here they are harvested and used as sensitive and specific detection tools that bind the protein directly.
After blocking, a dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL) is incubated with the membrane under gentle agitation. Typically, the solution is composed of buffered saline solution with a small percentage of detergent, and sometimes with powdered milk or BSA. The antibody solution and the membrane can be sealed and incubated together for anywhere from 30 minutes to overnight. It can also be incubated at different temperatures, with warmer temperatures being associated with more binding, both specific (to the target protein, the "signal") and non-specific ("noise").
Secondary antibody
After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to another antibody, directed at a species-specific portion of the primary antibody. Antibodies come from animal sources (or animal sourced hybridoma cultures); an anti-mouse secondary will bind to almost any mouse-sourced primary antibody, which allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and provides far more consistent results. This is known as a secondary antibody, and due to its targeting properties, tends to be referred to as "anti-mouse," "anti-goat," etc. The secondary antibody is usually linked to biotin or to a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal.
Most commonly, a horseradish peroxidase-linked secondary is used to cleave a chemiluminescent agent, and the reaction product producesluminescence in proportion to the amount of protein. A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies bound to the blot. A cheaper but less sensitive approach utilizes a 4-chloronaphthol stain with 1% hydrogen peroxide; reaction of peroxide radicals with 4-chloronaphthol produces a dark brown stain that can be photographed without using specialized photographic film.
As with the ELISPOT and ELISA procedures, the enzyme can be provided with a substrate molecule that will be converted by the enzyme to a colored reaction product that will be visible on the membrane (see the figure below with blue bands).
Another method of secondary antibody detection utilizes a near-infrared (NIR) fluorophore-linked antibody. Light produced from the excitation of a fluorescent dye is static, making fluorescent detection a more precise and accurate measure of the difference in signal produced by labeled antibodies bound to proteins on a Western blot. Proteins can be accurately quantified because the signal generated by the different amounts of proteins on the membranes is measured in a static state, as compared to chemiluminescence, in which light is measured in a dynamic state.[7]
A third alternative is to use a radioactive label rather than an enzyme coupled to the secondary antibody, such as labeling an antibody-binding protein like Staphylococcus Protein A or Streptavidin with a radioactive isotope of iodine. Since other methods are safer, quicker, and cheaper, this method is now rarely used; however, an advantage of this approach is the sensitivity of auto-radiography based imaging, which enables highly accurate protein quantification when combined with optical software (e.g. Optiquant).
Historically, the probing process was performed in two steps because of the relative ease of producing primary and secondary antibodies in separate processes. This gives researchers and corporations huge advantages in terms of flexibility, and adds an amplification step to the detection process. Given the advent of high-throughput protein analysis and lower limits of detection, however, there has been interest in developing one-step probing systems that would allow the process to occur faster and with less consumables. This requires a probe antibody which both recognizes the protein of interest and contains a detectable label, probes which are often available for known protein tags. The primary probe is incubated with the membrane in a manner similar to that for the primary antibody in a two-step process, and then is ready for direct detection after a series of wash steps.
Western blot using radioactive detection system
After the unbound probes are washed away, the Western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all Westerns reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin, that should not change between samples. The amount of target protein is indexed to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
The colorimetric detection method depends on incubation of the Western blot with a substrate that reacts with the reporter enzyme (such asperoxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
Chemiluminescent detection methods depend on incubation of the Western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which capture a digital image of the Western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used.
Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the Western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest (see image to the right). The importance of radioactive detections methods is declining[citation needed], because it is very expensive, health and safety risks are high, and ECL (enhanced chemiluminescence) provides a useful alternative.
The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera equipped with appropriate emission filters which captures a digital image of the Western blot and allows further data analysis such as molecular weight analysis and a quantitative Western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.
One major difference between nitrocellulose and PVDF membranes relates to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. While there are well-established protocols available for stripping nitrocellulose membranes, the sturdier PVDF allows for easier stripping, and for more reuse before background noise limits experiments. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.