Information about Western Blotting

A western blot (alternately, immunoblot) is a method to detect a specific protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) (Figure 1) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialise in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. This has dramatically reduced the time to carry out a blot. Previously large animals (e.g. sheep, goat - lots of serum) had to be immunised with the target protein twice (secondary immune response generates high affinity antibodies). Then either serum could be purified and used (polyclonal antibodies) or B cells could be isolated from the animal and fused in vitro with mouse myeloma cells to generate hybridomas that then provided single-specificity antibodies (monoclonal antibodies). Commercial antibodies are expensive, though the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, biochemistry, immunogenetics and other molecular biology disciplines.

Other related techniques include using antibodies to detect proteins in tissues and cells by immunostaining and enzyme-linked immunosorbent assay (ELISA).

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette^[1] 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

Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods.

Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes.

A combination of biochemical and mechanical techniques – including various types of filtration and centrifugation – can be used to separate different cell compartments and organelles.

Gel electrophoresis

Main article: Gel electrophoresis

The proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pI), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel.

By far the most common type of gel electrophoresis employs polyacrylamide gels and buffers loaded with sodium dodecyl sulfate (SDS). SDS-PAGE (SDS polyacrylamide gel electrophoresis) maintains polypeptides in a denatured state once they have been treated with strong reducing agents to remove secondary and tertiary structure (e.g. S-S disulfide bonds to SH and SH) and thus allows separation of proteins by their molecular weight. Sampled proteins become covered in the negatively charged SDS and move to the positively charged electrode through the acrylamide mesh of the gel. Smaller proteins migrate faster through this mesh and the proteins are thus separated according to size (usually measured in kilo Daltons, kD). The concentration of acrylamide determines the resolution of the gel - the greater the acrylamide concentration the better the resolution of higher molecular weight proteins. Proteins travel only in one dimension along the gel for most blots.

Samples are loaded into wells in the gel. One lane is usually reserved for a marker or ladder, a commercially available mixture of proteins having defined molecular weights, typically stained so as to form visible, coloured bands. An example of a ladder is the GE Full Range Molecular weight ladder (Figure 1). When voltage is applied along the gel, proteins migrate into it at different speeds. These different rates of advancement (different electrophoretic mobilities) separate into bands within each lane.

It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. 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 on top of the gel, and a stack of tissue papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it. Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane. The proteins have now moved 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.

The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie or Ponceau S dyes. Coomassie is the more sensitive of the two, although Ponceau S's water solubility makes it easier to subsequently destain and probe the membrane as described below.

Blocking

Since the membrane has been chosen for its ability to bind protein, and 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 Bovine serum albumin (BSA) or non-fat dry milk (both are inexpensive), with a minute percentage of detergent such as Tween 20. 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.

Detection

During the detection process the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reported enzyme, which when exposed to an appropriate substrate drives a colorimetric 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.

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, 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 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, the membrane 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. Antibodies come from animal sources (or animal sourced hybridoma cultures); an anti-mouse secondary will bind to just about any mouse-sourced primary antibody. This allows some cost savings by allowing an entire lab to share a single source of mass-produced antibody, and provides far more consistent results. 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 enhances the 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 method is now rarely used.

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 color 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 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.

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 95% ethanol, isopropanol or methanol before use. PVDF membranes also tend to be thicker and more resistant to damage during use.

2-D Gel Electrophoresis

2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. In the first dimension polypeptides are separated according to isolelectric point. In the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine) and negatively (e.g. glutamate) amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point.

In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.

Please refer to reference articles for examples of the application of 2-D SDS PAGE.

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 indicate 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.

Protocols

Dr. Mark Barton Frank Lab protocol

References

• Renart J, Reiser J, Stark GR. "Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure", Proc Natl Acad Sci U S A, 1979 Jul;76(7):3116-20. PMID: 91164 abstract
• Towbin H, Staehelin T, Gordon J. "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications" Proc Natl Acad Sci U S A, 1979 Sep;76(9):4350-4. PMID: 388439 abstract
• Citation's Classic: Burnette

See also

• Far-western blotting
• SDS-page

Related links

• Western blot analysis of sub-cellular fractionated samples using the Odyssey Infrared Imaging System (a protocol)
• Western blot protocol including example buffers and reprobing procedure

•  • [ e] Proteins: key methods of study
Experimental	Protein purification - Green fluorescent protein - Western blot - Protein immunostaining - Protein sequencing - Gel electrophoresis/Protein electrophoresis - Protein immunoprecipitation - Peptide mass fingerprinting
Bioinformatics	Protein structure prediction - Protein-protein docking - Protein structural alignment - Protein ontology - Protein-protein interaction prediction
Assay	Enzyme assay - Protein assay - Secretion assay

•  • [ e] Molecular probes
Southern blot (DNA) - Western blot (protein) - Northern blot (RNA) - Southwestern blot (protein:DNA) - Far-western blotting (protein:protein)