Western blot

A western blot (a.k.a immunoblot) is a method in molecular biology/biochemistry/immunogenetics to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane (typically nitrocellulose), where they are "probed" using antibodies specific to the protein. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups. Other techniques also using antibodies allow detection of proteins in tissues (immunohistochemistry) and cells (immunocytochemistry).

The name western blot was given to the technique by W. Neal Burnette (Analytical Biochemistry, 112:195-203, 1981) and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.

Steps in a western blot

Tissue preparation

Typically, samples are taken from either tissue or from cell culture. The samples are cooled or frozen rapidly. They are homogenized using sonication or mechanical force or simply lysed using high salt buffers (150mM). The resulting "whole-cell homogenate" or "whole-cell fraction" can be used as is, or subjected to centrifugation in a series of steps to isolate cytosolic (cell interior), nuclear and membrane fractions. The prepared sample is then assayed for protein concentration so that a consistent amount of protein can be taken from each different sample.

Samples are boiled from one to five minutes in a buffer solution (e.g. Laemmli's buffer - known as "sample buffer"), containing dye, a sulfurous compound - typically beta-mercaptoethanol or DTT, a detergent known as sodium dodecyl sulfate or SDS and glycerol. The boiling denatures the proteins, unfolding them completely. The SDS then surrounds the protein with a negative charge and the beta-mercaptoethanol prevents the reformation of disulfide bonds. The glycerol is neutraly charged in the stacking gel (as a result of the "upper buffer" PH=), and then is positively charged as it enters the resolving gel (as a result of the "lower buffer" PH=) thereby bypassing the proteins and allowing the proteins to separate according to molecular weight.

Gel electrophoresis

The proteins of the sample are separated according to molecular weight using gel electrophoresis. Gels have various formulations depending on the lab, molecular weight of the proteins of interest, and buffers available. Polyacrylamide gels are most common. Since the proteins travel only in one dimension along the gel, samples are loaded side-by-side into "wells" formed in the gel. Proteins are separated by mass into "bands" within each "lane" formed under the wells. One lane is reserved for a "marker," or "ladder," a commercially available mixture of proteins having defined molecular weights.

It is also possible to use a 2-D gel which spreads the proteins from a single sample out in two dimensions and proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.

Transfer

In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or PVDF. The membrane is placed face-to-face with the gel, and current is applied to large plates on either side. The charged proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this "blotting" process, the proteins are exposed on a thin surface layer for detection (see below). Both varieties of membrane are chosen for their non-specific protein binding properties (i.e. binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings.

Blocking

Since the membrane has been chosen for its ability to bind protein, steps must be taken to prevent non-specific protein interactions between it 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 Bovine serum albumin (BSA) or non-fat dry milk, with a minute percentage of detergent such as Tween 20 or colloidal carbon.

Detection

During the detection process the membrane is "probed" for the protein of interest with antibodies, and links them to a reporter enzyme, which drives a colorimetric or photometric signal. 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.

Two step

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 a part of the immune response, here they are harvested and used as sensitive and specific detection tools that bind the protein directly - hence "primary" antibody.

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 comprised 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, it is exposed to another antibody, directed at a species-specific portion of the primary antibody. 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 step confers an advantage in that several secondary antibodies will bind to one primary antibody, providing enhanced signal.

Most commonly, a horseradish peroxidase-linked secondary is used in conjunction with a chemiluminescent agent, and the reaction product produces luminescence 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.

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).

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 with a radioactive isotope of iodine. Since other methods are safer, quicker, and cheaper, this has fallen into disuse.

One step

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.

Analysis

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.

Colorimetric detection

The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different colour that precipitates next to the enzyme and thereby stains the nitrocellulose 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.

Chemiluminescence

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 captures 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. So-called "enhanced chemiluminescent" (ECL) detection is considered to be among the most sensitive detection methods for blotting analysis.

Radioactive detection

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, because it is very expensive, health and safety risks are high and ECL provides a useful alternative.

Fluorescent detection

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 which appropriate emission filters which captures a digital image of the western blot and allows further data analysis such molecular weight analysis and a quantitative western blot analysis. Fluorescence is considered to be among the most sensitive detection methods for blotting analysis.

Secondary probing

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 100% methanol or isopropanol before using. PVDF membranes also tend to be thicker and much more resistant to damage incurred by normal manipulation.

Medical diagnostic applications

The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indictate the proteins to which the patient's serum contains antibody.
A Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease').
Some forms of Lyme disease testing employ Western blotting.

External links

Western Blotting Information and Protocols

Protein methods | Molecular biology | Biochemistry | Laboratory techniques

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