Immunoblotting and western blotting Western blotting (immunoblot): Gel electrophoresis for proteins Hint: Browse through almost 875,000 products for Western blotting (immunoblot) Western Blotting (also called immunoblotting) is a technique used for analysis of individual proteins in a protein mixture (e.g. a cell lysate). In Western blotting (immunoblotting) the protein mixture is applied to a gel electrophoresis in a carrier matrix (SDS-PAGE, native PAGE, isoelectric focusing, 2D gel electrophoresis, etc.) to sort the proteins by size, charge, or other differences in individual protein bands. The separated protein bands are then transferred to a carrier membrane (e.g. nitrocellulose, nylon or PVDF). This process is called blotting. The proteins adhere to the membrane in the same pattern as they have been separated due to interactions of charges. The proteins on this immunoblot are then accessible for antibody binding for detection. CONTENTS Western blotting (immunoblot) setup Types of gel electrophoresis Blotting Immunodetection Why western blotting? Antibodies are used to detect target proteins on the western blot (immunoblot). The antibodies are conjugated with fluorescent or radioactive labels or enzymes that give a subsequent reaction with an applied reagent, leading to a coloring or emission of light, enabling detection. The term Western blotting is based on a play of words. The southern blot, which is a method to detect specific DNA sequences, is named after Ed Southern, who first described this procedure. The western blot (immunoblot), as well as the northern blot (for RNA detection), play on the meaning of this name. Go back to top If you are interested in our newsletter, follow the button below to subscribe. To see a newsletter example, please follow this link. Subscribe to our newsletter What type of gel electophoresis for proteins are possible? One can choose from different types of gel electrophoresis for proteins depending on the criteria by which the proteins should be separated. Some commonly used electrophoretic methods are: SDS-PAGE, native-PAGE and isoelectric focusing. SDS-PAGE: This is a denaturing method as it treats the proteins with anionic SDS detergent (sodiumdodcylsulfate). Secondary- and tertiary structure are destroyed by this process. Additionally, SDS binds the proteins and thereby covers their chemical charges, leading to equally negatively charged proteins. Therefore the following separation happens solely by the size of the polypeptide chains in the polyacrylamide gel. Native PAGE: Native, unfolded, and not-denatured proteins can be separated using this method. This method allows for the separation of proteins that are inaccessible by other methods. One example would be the separation of modified and unmodified proteins of the same kind (e.g. phosphorylated versus unphosphorylated state of a protein). Native PAGE can also be used to confirm biologically relevant conformations, like di-, tri-, or tetrameric forms of proteins (contrary to SDS-PAGE, which would separate the individual and denatured peptide chains). This method can also detect different complexes of different proteins. The separation using native PAGE depends on a number of parameters such as the charge, size and 3D structure of the protein. A suitable buffer is needed to maintain the 3D folding of the protein. The applicability of the buffer depends on the isoelectric point and the charges of the protein. Isoelectric focusing: This method builds on the fact that a protein has a specific charge at certain pH values. Depending on the pH the acidic and basic functional groups contribute by increasing or decreasing the total charge of the protein. The isoelectric point is defined as the the point where the total charge of the molecule is zero, because there is an equal amount of negative and positive charges in the molecule. Special gradient gels are needed for isoelectric focusing as the pH changes from acidic to basic along a gradient within the gel. Due to an electric charge connected to the gel the protein travels to the point in the gel where the charge of the gel equals that of the protein, and the total charge equals zero, i.e. the isoelectric point. Hence, this method is used to separate proteins by their charges, as well as to determine the isoelectric point of a target protein. The separation occurs due to the charge of the protein or by the number of basic- and acidic groups the protein contains. The above-mentioned methods for gel electrophoresis of proteins can also be combined to separate proteins. The choice of methods depends on the specific requirements of the experiment. Go back to top Blotting Following the separation of the protein mix the polypeptide bands are transferred to a membrane carrier. For this purpose the membrane is attached to the gel and this so-called sandwich is transferred to an electrophoresis chamber. It is possible that some of the SDS is washed out, and the protein partially re-naturates again, i.e. regains its 2D- and 3D structure. However, the applied electric charge causes the proteins to travel out of the gel vertically to the direction they traveled in on the gel, onto the membrane. The protein bands are thereby bound to the membrane. The "blotted" bands are now available to be treated further (e.g. for detection of specific proteins with specific antibodies). Hint: Loading controls are essential to assure comparable amounts of protein in different samples as well as the homogenous protein transfer to the membrane during the blotting procedure. Go back to top Immunodetection The identification of specific antibodies is possible after the separation and blotting of proteins. Specific antibodies (mono- or polyclonal) bind to "their" band of proteins. Unspecifically binding antibodies are removed by washing with detergent-containing buffers. Additionally, unspecific binding pockets can be blocked before the addition of specific antibodies. Primary antibodies are usually applied first, which are then recognized by a secondary antibody. The secondary antibody is conjugated with colour, radioactivity or an enzyme for detection. Biotin-conjugated antibodies are also used for this purpose. It can occasionally be advantageous to use polyclonal primary antibodies as such antibodies recognize several epitopes, contrary to monoclonal antibodies that are restricted in their binding affinity. After immunodetection it is possible to strip the antibody off the membrane for further analysis with other antibodies (e.g. in order to detect other specific antibodies from the protein mixture under investigation). Analysis of the western blot is then carried out using a variety of different imaging systems (e.g. luminescence, color reaction, autoradiography). Hint: The identification of a protein of interest in a Western blot relies among other factors on its molecular weight. To this end, the electrophoretically separated proteins in the sample are compared with a molecular weight standard of a known composition (see here and here). Go back to top Why western blotting (immunoblot)? The western blotting (immunoblot) method entails various advantages as compared to other immunosorbent assays (ISAs), like for example ELISA. Western blotting (immunoblot) expands on the idea of ELISA by allowing separation of the protein mix by size, charge, and/or conformation. The described method of stripping allows for the detection of several targets, contrary to ELISA where only one protein can be detected. As the gel electophoreis of proteins separates the proteins into bands, one can determine the size of the target protein/polypeptide. It is also possible to (semi-)quantify the protein of interest by running an internal quantity standard in parallel with the samples in the gel. Similarly, the protein content of the samples can be compared ("sample A contains more protein than sample B"). A disadvantage of western blotting (immunoblot) is that it is time-consuming (compared to ELISA) and has a high demand in terms of experience of the experimenter. Additionally, it requires optimizing the experimental conditions (i.e. protein isolation, buffers, type of separation, gel concentration, etc.). There are many different types and methods for western blotting (immunoblot). Hence, it covers very different topics and applications.