Overview of Western Blotting
The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis. After electrophoresis, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. Most commonly, the transferred protein is complexed with an enzyme-labeled antibody as a probe. An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic precipitate on the membrane for colorimetric detection. The most sensitive detection methods use a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct. The light output can be captured using film, a CCD camera or a phosphorimager that is designed for chemiluminescent detection. Alternatively, fluorescently tagged antibodies can be used, which are directly detected with the aid of a fluorescence imaging system. Whatever system is used, the intensity of the signal should correlate with the abundance of the antigen on the membrane.
Detailed procedures for detection of a Western blot vary widely. One common variation involves direct vs. indirect detection. With the direct detection method, the primary antibody that is used to detect an antigen on the blot is labeled with an enzyme or fluorescent dye. This detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons. In the indirect detection method, a primary antibody is added first to bind to the antigen. This is followed by a labeled secondary antibody that is directed against the primary antibody. Labels include biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme conjugates such as horseradish peroxidase or alkaline phosphatase. The indirect method offers many advantages over the direct method.
Western Blotting Handbook and Troubleshooting Guide
The "Thermo Scientific Pierce Western Blotting Handbook and Troubleshooting Guide" (2014) details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer, including Pierce Fast Western products. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide.
- Quicker since only one antibody is used
- No concern for cross-reactivity of a secondary antibody
- Double possible with different labels on primary antibodies
- Labeling may reduce immunoreactivity of primary antibody
- Labeled primary antibodies are expensive
- Low flexibility in choice of primary antibody label
- Little signal amplification
- Secondary antibody can amplify signal
- A variety of labeled secondary antibodies are available
- One secondary may be used with many primary antibodies
- Labeling does not affect primary antibody immunoreactivity
- Changing secondary allows change of detection method
- Secondary antibodies may produce nonspecific staining
- Additional steps required compared to the direct method
Comparison of direct and indirect Western blot detection methods.
Electrophoretic Separation of Proteins
Gel electrophoresis is a technique in which charged molecules, such as protein or DNA, are separated according to physical properties as they are forced through a gel by an electrical current. Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins within a single sample. When combined with Western blotting, PAGE is a powerful analytical tool providing information on the mass, charge, purity or presence of a protein. Several forms of PAGE exist and can provide different types of information about the protein(s).
Transfer Proteins to a Membrane
Following electrophoresis, the protein must be transferred from the electrophoresis gel to a membrane. There are a variety of methods that have been used for this process, including diffusion transfer, capillary transfer, heat-accelerated convectional transfer, vacuum blotting transfer and electroelution. The transfer method that is most commonly used for proteins is electroelution or electrophoretic transfer because of its speed and transfer efficiency. This method uses the electrophoretic mobility of proteins to transfer them from the gel to the membrane. Electrophoretic transfer of proteins involves placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose or other suitable, protein-binding support and "sandwiching" this between two electrodes submerged in a conducting solution. When an electric field is applied, the proteins move out of the polyacrylamide gel and onto the surface of the membrane, where the proteins become tightly attached. The result is a membrane with a copy of the protein pattern that was originally in the polyacrylamide gel.
Transfer efficiency can vary dramatically among proteins, based upon the ability of a protein to migrate out of the gel and its propensity to bind to the membrane under a particular set of conditions. The efficiency of transfer depends on factors such as the composition of the gel, complete contact of the gel with the membrane, the position of the electrodes, the transfer time, size and composition of proteins, field strength and the presence of detergents and alcohol in the buffer.
After transfer and before proceeding with the Western blot, total protein on the membrane is often stained with a dye, such as Ponceau S or amido black 10B, to check the transfer efficiency; the gel may also be stained to confirm that protein has been moved out of the gel, but this does not ensure efficient binding of protein to the membrane. Because dyes may interfere with antibody binding and detection, a protein stain that is easily removable is ideal. Ponceau S stain is the most widely used reagent for reversibly staining proteins on a membrane, although it has limited sensitivity, does not photograph well and fades quickly, which makes documentation difficult. Superior alternatives for staining protein on nitrocellulose or PVDF membranes are available, which allow the detection of low-nanogram levels of protein, are easily photographed and do not fade until removed.
Blocking Nonspecific Sites
The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block free sites on a membrane. The blocking buffer should improve the sensitivity of the assay by reducing background interference and improving the signal to noise ratio. No single blocking agent is ideal for every occasion since each antibody-antigen pair has unique characteristics. For true optimization, empirical testing of blocking buffers is essential.
Wash Buffer Formulations
Like other immunoassay procedures, Western blotting consists of a series of incubations with different immunochemical reagents separated by wash steps. Washing steps are necessary to remove unbound reagents and reduce background, thereby increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot. As with other steps in performing a Western blot, a variety of buffers may be used.
Occasionally, wash buffer formulations consist of only a physiological buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) without any additives. More commonly, a detergent such as 0.05% Tween* 20 is added to the buffer to help remove nonspecifically bound material. Depending on the specifics of the assay, the amount of detergent in the wash buffer will vary, though typical concentrations are from 0.05 to 0.5% for detergents like Tween 20. Another common technique is to add a 1:10 dilution of the blocking solution to the wash buffer. Including the blocking agent with the detergent may help to minimize background in the assay by preventing elution of the blocking protein from the membrane and/or allowing nonspecific interactions to occur with the protein in solution rather than those immobilized on the membrane.
It is important to note that detergents, like the protein solutions, can promote microbial growth. While it is convenient to make pre-diluted stocks of detergents like NP-40, CHAPS, and Tween 20, fungi can grow in these solutions which can lead to high background noise. In addition, detergents can contain significant amounts of peroxides which will cause background signal when using horseradish peroxidase substrates. Therefore, it is important to use high-purity detergents.
Primary and Secondary Antibodies
Although other methods are used, Western blotting is typically performed by probing the blocked membrane with a primary antibody that recognizes a specific protein or epitope on a group of proteins (e.g., SH2 domain or phosphorylated tyrosine). The choice of a primary antibody for a Western blot will depend on the antigen to be detected and what antibodies are available to that antigen. It is also important to note that not all primary antibodies are suitable for Western blotting and the feature should be verified, if possible, before purchasing a new primary antibody.
In general, the primary antibody which recognizes the target protein in a Western blot is not directly detectable. Therefore, tagged secondary antibodies or other detection reagents are used as the means of ultimately detecting the target antigen (indirect detection). A wide variety of labeled secondary detection reagents can be used for Western blot detection. The choice of which depends upon either the species of animal in which the primary antibody was raised (the host species) or any tag on that antibody (i.e., biotin or DIG). For example, if the primary antibody is an unmodified mouse monoclonal antibody then the secondary antibody must be an anti-mouse IgG secondary antibody obtained from a non-mouse host.
Antibodies for Western blotting are typically used as dilute solutions, ranging from a 1/100-1/500,000 dilution from a 1mg/mL stock solution. The optimal dilution of a given antibody with a particular detection system must be determined experimentally. More sensitive detection systems require less antibody than lower sensitivity systems and can result in substantial savings on antibody costs and allow a limited supply of antibody to be stretched out over more experiments. Using lower amounts of antibody can also have the added benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity.
Antibody dilutions are typically made in the wash buffer. The presence of detergent and a small amount of the blocking agent in the antibody diluent often helps to minimize background thereby increasing the signal:noise ratio. Conversely, adding too much blocking agent or detergent to the antibody dilution solution can prevent efficient binding of the antibody to the antigen, causing reduced signal as well as reduced background.
Watch this video on probing the blot for your protein of interest
While there are many different tags that can be conjugated to a secondary or primary antibody, the detection method used will limit the choice of what can be used in a Western blotting assay. Radioisotopes were used extensively in the past, but they are expensive, have a short shelf-life, offer no improvement in signal:noise ratio and require special handling and disposal. Alternative labels are enzymes and fluorophores.
Enzymatic labels are most commonly used for Western blotting and, although they require extra steps, can be extremely sensitive when optimized with an appropriate substrate. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates are available for use with either enzyme. Alkaline phosphatase offers a distinct advantage over other enzymes in that its reaction rate remains linear allowing sensitivity to be improved by simply allowing a reaction to proceed for a longer time period. Unfortunately, the increased reaction time often leads to high background signal resulting in low signal:noise ratios. Horseradish peroxidase (HRP) conjugated antibodies are considered superior to antibody-AP conjugates with respect to the specific activities of both the enzyme and antibody due the smaller size of HRP enzyme and compatibility with conjugation reactions. In addition, the high activity rate, good stability, low cost and wide availability of substrates make HRP the enzyme of choice for most applications.
Enzyme conjugated antibodies offer the most flexibility in detection and documentation methods for Western blotting because of the variety of substrates available. The simplest detection/documentation system is to use chromogenic substrates. While not as sensitive as other substrates, the chromogenic substrates allow direct visualization of blot development. Unfortunately, chromogenic substrates tend to fade as the blot dries or when stored making the blot itself an unreliable means of documentation. However, it is fairly straightforward to either photocopy or directly scan the blot in order to make a permanent replica of chromogenic Western blot results.
Chemiluminescent blotting substrates differ from other substrates in that the signal is a transient product of the enzyme-substrate reaction and persists only as long as the reaction is occurring. If either the substrate is used up or the enzyme loses activity then the reaction will cease and signal will be lost. However, in well-optimized assays using proper antibody dilutions and sufficient substrate, the reaction can produce stable output of light for 1 to 24 hours depending on the substrate, allowing consistent and sensitive detection that may be documented with X-ray film or digital imaging equipment. While X-ray film can be used to obtain semi-quantitative data, digital imaging is more sensitive because of the broad dynamic range of detection, allowing researchers to obtain quantitative data from Western blots.
The use of fluorophore-conjugated antibodies in an immunoassays requires fewer steps because there is no substrate development step in the assay. While the protocol is shorter, this method requires special equipment in order to detect and document the fluorescent signal due to the need for an excitation light source. Recent advances in digital imaging and develop of new fluorophores such as infrared, near-infrared and quantum dots has increased the sensitivity and popularity of using fluorescent probes for Western blotting and other immunoassays. Although the equipment and fluorescent-conjugated antibodies can be quite expensive, this method has the added advantage of multiplex compatibility (using more than one fluorophore in the same experiment). In addition, chemical waste is further reduced compared to other blotting procedures.
For Research Use Only. Not for use in diagnostic procedures.