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Since its introduction in 1979 (Towbin et al., 1979), protein blotting has become a routine tool in research laboratories. It is traditionally used to detect low amounts of proteins in complex samples or to monitor protein expression and purification. The simplest protein blotting procedure, known as dot blot or slot blot, uses vacuum filtration to transfer protein onto a microporous membrane. While this method may provide qualitative information about total protein expression levels and can be performed on multiple samples in parallel, it lacks information on protein molecular weight. Also, specificity can be compromised as protein degradation products or post-translationally- modified isoforms may be detected along with the intact protein.
A more complex procedure, Western blotting, involves the separation of a protein mixture by gel electrophoresis, with subsequent electrotransfer to a suitable membrane (e.g., PVDF). A protein can be identified through its reaction with a specifically labeled antibody or antigen. See “Membrane-based Immunodetection” figure. Through spatial resolution this method provides molecular weight information on individual proteins and separates isoforms from processed products. After proteins have been transferred onto a suitable membrane, they can be stained for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection.
In the clinical laboratory, immunoblotting has emerged for applications in fields such as infectious and autoimmune diseases, allergy, and others (Towbin et al., 1989; Stahl et al., 2000). Western blotting is considered to be a reliable confirmatory diagnostic test following a repeatedly reactive ELISA over the course of viral infection, and is reported to be the most sensitive, unequivocal and simple test system available, with the highest complexity of information obtained (Bauer, 2001; Mylonakis et al., 2000; Heermann et al., 1988).
Examples of Western blotting applications include analysis of protein expression in yeast by quantitative western analysis (Ghaemmadami et al., 2003), determination of protein copy number and compartmentalization (Rudolph et al., 1999), study of competitive protein kinase inhibition by ATP (Wang and Thompson, 2001), and detection of genetically modified organisms in crops and foods (Ahmed, 2002).
In addition, new blotting techniques and applications are being developed. A “double blotting” method (Lasne, 2001, 2003) eliminates false positives due to strong non-specific interactions between the blotted proteins and unrelated secondary antibodies.
When developing new immunodetection protocols, all components and their interactions must be considered. Antibody concentrations, buffer compositions, blocking agents and incubation times must be tested empirically to determine the best conditions. Water quality is important in all steps - small impurities can cause big problems. For instance, the enzyme activity of horseradish peroxidase is inhibited by pyrogens, a common contaminant of even high purity water, and azide, a common preservative in antibody solutions.
The quality of the blocking agents must also be considered relative to consistency and contaminants.
The following sections provide important information regarding immunodetection. Understanding these basic concepts will help to optimize protocols for specific samples.
The two most commonly used buffers are phosphate buffered saline (PBS) and Tris-buffered saline (TBS). Many variations on the compositions of these buffers Membrane-based have been published. The key feature is that the buffer must preserve the biological activity of the antibodies. Thus, the ionic strength and pH should be fairly close to physiological conditions. PBS formulations with 10 mM total phosphate concentration work well with a wide array of antibodies and detection substrates.
While incubating, the container holding the membrane should be gently agitated. A sufficient volume of buffer should be used to cover the membrane completely so that it is floating freely in the buffer. If more than one blot is placed in a container, insufficient buffer volume will cause the blots to stick together. This will limit the accessibility of the incubation solutions and can cause a variety of artifacts including high backgrounds, weak signals, and uneven sensitivity.
For meaningful results, the antibodies must bind only to the protein of interest and not to the membrane. Non-specific binding (NSB) of antibodies can be reduced by blocking the unoccupied membrane sites with an inert protein or non-ionic detergent. The blocking agent should have a greater affinity for the membrane than the antibodies. It should fill all unoccupied binding sites on the membrane without displacing the target protein from the membrane. The most common blocking agents used are bovine serum albumin (BSA, 0.2-5.0%), non-fat milk, casein, gelatin, and dilute solutions of Tween®-20 detergent (0.05-0.1%). Tween-20 detergent was also shown to have a renaturing effect on antigens, resulting in improved recognition by specific antibodies (Van Dam et al., 1990; Zampieri et al., 2000). Other detergents, such as Triton® X-100 detergent, SDS, and NP-40, are sometimes used but can be too harsh and disrupt interaction between proteins.
The blocking agent is usually dissolved in PBS or TBS buffers.
There are risks associated with blocking; a poorly selected blocking agent or excessive blocking can displace or obscure the protein of interest.Therefore, the correct choice of a blocking agent can be critical to a successful immunodetection. For example, dry milk cannot be used with biotinylated or concanavalin-labeled antibodies, since milk contains both glycoproteins and biotin. The analysis of phosphorylated proteins with phospho-specific antibodies can be compromised if using crude protein preparation as a blocking agent. These preparations may contain phosphatases, and the phosphorylated proteins on the blot could become dephosphorylated by this enzyme. It has been shown that addition of phosphatase inhibitors to the blocking solution increases the signal with phospho- specific antibody (Sharma and Carew, 2002). Finally, a blocking agent that is found to be suitable for one antigen-antibody combination may not be suitable for another.
Compatibility between the blocking agent and the detection reagent can be determined easily using the following Spot Blotting Method. The blocking solution gets spotted onto a blank PVDF membrane that has been wet in methanol and equilibrated in TBS. Detection reagents are then added to the blot, the blot is incubated for 5 minutes and then exposed to X-ray film. Appearance of a dark spot indicates that the blocking reagent is incompatible with the detection reagent. It is important to remember that Immobilon-PSQ transfer membrane, with its higher surface area and smaller pore size than Immobilon-P transfer membrane, binds more protein. If Immobilon- PSQ transfer membrane is substituted directly for Immobilon-P transfer membrane in a standard Western blotting procedure, there may be insufficient blocking reagent to saturate the membrane surface. Additional washing steps may also be required to reduce the background. Blocking can be conveniently optimized using the Spot Blotting Method described earlier.
After blocking, the blot is incubated with one or more antibodies. The first antibody binds to the target protein, and a secondary antibody binds to the first. The secondary antibody is conjugated to an enzyme or dye that is used to indicate the location of the protein.
Although the primary antibody may be labeled directly, using a secondary antibody has distinct advantages. First, more than one molecule of the secondary antibody may be able to bind to a single molecule of the primary antibody, resulting in signal amplification. Second, a labeled secondary antibody (enzyme-antibody conjugate) can be used for a large number of primary antibodies of different specificities, thereby eliminating the need to label numerous primary antibodies.
Either polyclonal or monoclonal antibodies are used. Polyclonal antibodies usually come in the form of antiserum or affinity-purified antibody. Monoclonal antibodies are expressed in ascites fluid or tissue culture fluid and can be directly used or as an affinity- purified preparation. It is important to remember that a denatured protein may not be recognized by an antibody raised to the native antigen. In some cases, a nondenaturing gel may be required for production of the blot. Antibodies are diluted in buffer and blocking solution to prevent non-specific binding to the membrane.
The antibody diluent also normally contains trace amounts of Tween-20 or another detergent to prevent non-specific aggregation of the antibodies. Many published protocols for chemiluminescence require 0. 1% (v/v) Tween-20 in the blocking solution and antibody diluent. It is important to recognize that concentrations above 0.05% (v/v) have the potential to wash some blotted proteins from the membrane. Elevating the concentration of Tween-20 detergent is often used to reduce the background. Often, a simpler and more cost-effective strategy is to reduce the concentration of the antibodies, notably the secondary antibody (see “Optimization of secondary antibody dilution” figure below).
In addition to being specific for the protein of interest, the antibodies must not cross-react with components of the blocking buffer and should be relatively pure. Impurities in the form of other proteins or aggregates can result in nonspecific binding and increased background.
Immunodetection is an extremely sensitive method. In order to achieve a high signal-to-noise ratio and thereby maximum sensitivity, the concentration of primary and secondary antibodies should be optimized for each case. Generally, non-specific signal can be alleviated by higher dilution of the primary antibody or decreased protein load on the original gel. High overall background can be minimized by higher dilution of the enzyme conjugated secondary antibody.
Optimal concentration of both primary and secondary antibodies also depends on the sensitivity of the detection reagents. Up to twenty times less antibody is required for high-sensitivity reagents (detection at femtogram level) as compared to low- sensitivity reagents (detection at the picogram level).
Washing the blot removes any unbound antibodies from the membrane that could cause high background and poor detection. A dilute solution of Tween-20 (0.05% v/v) in PBS or TBS buffer is commonly used, especially when the antibody preparations are comparatively crude or used at high concentrations. As mentioned previously, higher concentration detergent solutions could elute the protein of interest from the membrane. For highly purified antibodies, buffer alone is often sufficient for washing.
The amount of washing required is best determined experimentally. Too little washing will yield excessive background, while overwashing may elute the antibodies and reduce the signal. It is recommended that washing be performed a minimum of three times for 5 minutes each.
Persistent background can be reduced by adding up to 0.5M sodium chloride and up to 0. 2% SDS to the TBS wash buffer and extending wash time to 2 hours.
An innovative method to eliminate non-specific binding in Western blots was developed by Dr. Francoise Lasne of the Laboratoire National de Depistage du Dopage (National Anti-Doping Laboratory) in Chatenay-Malabry, France (Lasne, 2001; Lasne, 2003). Dr. Lasne has been working on recombinant human erythropoietin (EPO) detection.She has found that recombinant and naturally occurring EPO have different isoelectric points (pI). Recombinant EPO has a pI of 4.42?5.11, while natural EPO has a more acidic pI of 3.92?4.42. However, when urine samples are blotted, the very high non-specific binding (NSB) of the secondary antibodies makes it difficult to distinguish between recombinant and natural EPO. To eliminate the NSB, Dr. Lasne developed an innovative solution called "double blotting." After the primary antibody is bound to the blotted protein, the antibodies are transferred to a second Immobilon-P membrane under acidic conditions. The primary antibody desorbs from its corresponding antigen and transfers through an intermediate onto the second (double-blot) membrane. When the double-blot membrane is probed with the secondary antibody, no other proteins are present to bind non-specifically, thus eliminating the background problem.
“Double blotting” has also been used in the detection of transthyretin and may be a useful method in detecting other low concentration serum or urine proteins.
Modern immunodetection methods are based on enzyme-linked detection, utilizing secondary antibodies covalently bound to enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The conjugated enzyme catalyzes the degradation of specific substrates, resulting in signal generation. Three types of substrates are commonly used: chromogenic, chemiluminescent, and chemifluorescent, as well as detection with fluorophore-labeled secondary antibodies. Immobilon PVDF membranes have been tested for compatibility with all commercially available chromogenic and chemiluminescent substrates.
Chromogenic detection in the figure below uses the enzyme to catalyze a reaction resulting in the deposition of an insoluble colored precipitate, for example, the insoluble blue compound obtained through the interaction of 5-bromo-4-chloro-3indolylphosphate (BCIP) and nitroblue tetrazolium salt (NBT) (Leary, et al., 1983). This technique is easy to perform and requires no special equipment for analysis. However, the following should be kept in mind:
Chemiluminescent detection uses an enzyme to catalyze a reaction that results in the production of visible light. Some chemiluminescent systems are based on the formation of peroxides by horseradish peroxidase; other systems use 1,2-dioxetane substrates and the enzyme alkaline phosphatase (Cortese, 2002). This technique has the speed and safety of chromogenic detection at sensitivity levels comparable to radioisotopic detection. Detection is achieved by either exposing the blot to X-ray film or acquiring the image directly in a chemiluminescence- compatible digital imaging system, usually equipped with highly-cooled CCD cameras to avoid electronic noise. Reprobing is possible with chemiluminescent substrates.
There are a variety of chemiluminescent substrates offering researchers different levels of sensitivity of detection. Traditional or low-sensitivity substrates allow protein detection at the picogram level. While these substrates may be appropriate for routine applications, they cannot detect low abundance proteins. New, high-sensitivity substrates, such as Immobilon substrates, allow visualization of proteins at the femtogram level. However, use of these powerful substrates often requires optimization of primary and secondary antibody concentrations.
When switching from a low-sensitivity to a high-sensitivity substrate like Immobilon Western HRP substrate, it is recommended that antibody dilutions be increased to avoid excessive background and appearance of non-specific bands.
Reagents for ECL immunodetection can be prepared using p-iodophenol (PIP) and luminol (Hengen, 1997). PIP is needed for enhancing the visible light reaction by acting as a co-factor for peroxidase activity toward luminol. When phenolic enhancers are used in combination with HRP, the level of light increases about 100-fold (Van Dyke and Van Dyke, 1990). These homemade reagents are cited to produce excellent results, however, the highest purity of the luminol and PIP is critical (Hengen, 1997).
Fluorescent detection employs either a fluorophoreconjugated antibody or fluorogenic substrates that fluoresce at the site of enzyme activity (chemifluoresence). One advantage of this method is that the fluorescent signal is stable for long periods of time, and blots can be archived and re-imaged. In addition, the wide variety of fluorophores makes it possible to simultaneously detect multiple protein targets in a single sample (multiplex detection).
Until recently, fluorescent detection in Western blotting was limited by the high fluorescent background of most blotting membranes. Millipore’s Immobilon-FL transfer membrane exhibits low background fluorescence across a wide range of excitation/emission wavelengths compared to other blotting membranes (see “Reverse Image demonstrating fluorescent detection,” below ). The membrane is ideal for any application involving fluorescence based immunodetection, including chemifluorescent substrates and multiplexing (see “Example of fluorescence-based immunodetection,” below). In addition, Immobilon-FL membrane can be used for standard chemiluminescent or chromogenic detection.
A single blot can be sequentially analyzed with multiple antibodies by stripping the first antibody from the blot and incubating with another. This may be especially useful for co-localization experiments and method optimization or when sample amount is limited. Refer to Membrane Stripping Protocols.
The stripping process disrupts the antigen-antibody bonds and releases the antibody into the surrounding buffer. This is usually achieved either by a combination of detergent and heat or by exposure to low pH. Neither method removes the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TMB). However, it is still possible to analyze the blot with an antibody specific for a different target protein.