The western blot method is composed of
gel electrophoresis to separate native proteins by 3-D structure or
denatured proteins by the length of the polypeptide, followed by an electrophoretic transfer onto a membrane (mostly
PVDF or
nitrocellulose) and an immunostaining procedure to visualize a certain protein on the blot membrane.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) is generally used for the denaturing electrophoretic separation of proteins.
Sodium dodecyl sulfate (SDS) is generally used as a
buffer (as well as in the gel) in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged. Prior to electrophoresis, protein samples are often boiled to denature the proteins present. This ensures that proteins are separated based on size and prevents
proteases (enzymes that break down proteins) from degrading samples. Following electrophoretic separation, the proteins are transferred to a membrane (typically nitrocellulose or PVDF). The membrane is often then stained with
Ponceau S in order to visualize the proteins on the blot and ensure a proper transfer occurred. Next the proteins are blocked with milk (or other blocking agents) to prevent non-specific antibody binding, and then stained with
antibodies specific to the target protein. Besides that, the ideal lysis buffer is needed to acquire substantial amounts of target protein content because the buffer is leading the process of protein solubilization and preventing protein degradation. After completing the sample preparation, the protein content is ready to be separated by the utilization of gel electrophoresis. At least seven different approaches for total protein staining have been described for western blot normalization:
Ponceau S, stain-free techniques, Sypro Ruby,
Epicocconone,
Coomassie R-350,
Amido Black, and
Cy5.
Blocking 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 the 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) or I-Block, with a minute percentage (0.1%) of detergent such as
Tween 20 or
Triton X-100. Although non-fat dry milk is preferred due to its availability, an appropriate blocking solution is needed as not all proteins in milk are compatible with all the detection bands. or
epicocconone. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers. (see
western blot normalization)
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 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 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
CCD cameras which capture a digital image of the western blot or photographic film. The use of film for western blot detection is slowly disappearing because of non linearity of the image (non accurate quantification). 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 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 above). The importance of radioactive detections methods is declining due to its hazardous radiation , because it is very expensive, health and safety risks are high, and ECL (enhanced chemiluminescence) provides a useful alternative.
Fluorescent detection primary antibody and an
IR-dye labelled secondary antibody in
Leishmania major extracts The
fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as a 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 one of the best methods for quantification but is less sensitive than chemiluminescence.
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.
Minimum requirement specification for Western Blot In order to ensure that the results of Western blots are reproducible, it is important to report the various parameters mentioned above, including specimen preparation, the concentration of protein used for loading, the percentage of gel and running condition, various transfer methods, attempting to block conditions, the concentration of antibodies, and identification and quantitative determination methods. Many of the articles that have been published don't cover all of these variables. Hence, it is crucial to describe different experimental circumstances or parameters in order to increase the repeatability and precision of WB. To increase WB repeatability, a minimum reporting criteria is thus required. == 2-D gel electrophoresis ==