Western Blotting: Background information

Western Blotting is a technique, used for analysis of individual proteins in a protein mixture (e.g. a cell-lysate). Initially, the protein-mixture is applied to a gel-electrophoresis in a carrier matrix (SDS-PAGE, native-PAGE, isoelectric focusing, 2D-gelelectrophoresis, etc.) in order to sort the proteins by size, charge, or other differences in individual protein-bands. In a next step, the separated protein-bands are transferred to a carrier-membrane (e.g. nitro-cellulose, nylon or PVDF). This process is called blotting. Due to interactions of charges the proteins adhere to the membrane in the same pattern as they have been separated. The proteins are then accessible for antibody-binding in order to detect them.

For detecting the target proteins, antibodies are utilized that are for example 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.

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The name Western Blot is based on a play on words. The southern blot, which is a method for the detection of specific DNA-sequences, is named after Ed Southern, who first described this procedure. The western blot, as well as the northern blot (for RNA detection), play on the meaning of this name.

METHODOLOGY

Gel electrophoresis

Depending on the criteria by which the proteins should be separated one can choose from different types of electrophoresis. Frequently used 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. For instance in the case of modified and unmodified proteins of the same kind (e.g. phosphorylated versus unphosphorylated state of a protein). Importantly, it can be used in case of biologically relevant confirmations, like di-, tri-, or tetrameric forms of proteins (contrary to SDS-PAGE, which would separate the individual and denatured peptide chains). It is also possible to detect different complexes of different proteins.
The separation using native-PAGE depends on a number of parameters. Charge of the proteins, the size, and 3D structure. A suitable buffer is needed in order to maintain the 3D-folding of the protein. The applicability of the buffer depends on the isoelectric point, and the charges of the protein, respectively.

Isoelectric focusing: This method builds on the fact, that a protein has a specific charge at certain pH-values. Depending on the pH-value 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, i.e. the pH-Value 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.

In addition to the methods mentioned above, methods may also be combined in order to separate proteins. The choice of methods depends on the specific requirements of the experiment.

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).

Immunodetection

The identification of specific antibodies is possible after the separation and blotting of proteins has been carried out. 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.
Usually, primary antibodies are applied first, which are then recognized by a secondary antibody. The secondary antibody is conjugate with colour, radioactivity or an enzyme for detection. Also biotin-conjugated antibodies are in use.
It can occasionally be advantageous to use polyclonal primary antibodies, because such antibodies recognize several epitopes, contrary to monoclonal antibodies that are restricted in their binding affinity. After immuno-detection 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 (luminescent, color-reaction, autoradiography, and so on).

WHY WESTERN BLOTTING?

The western blotting method entails various advantages as compared to other immunosorbent assays (ISAs), like for example ELISA. The method 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 one protein only can be detected. Due to the fact that the separation into bands gives information about the size of the target protein/ polypeptide, (semi-)quantification is possible. To achieve this an internal quantity-standard is applied to the gel, in parallel with the samples. Similarly, the protein content of the samples can be compared ("sample A contains more protein than sample B"). A disadvantage of western blotting 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 of the experimental conditions (i.e. in terms of protein-isolation, buffers, type of separation, gel-concentration, etc.).

There are many different types, and methods for western blotting. Hence, it covers very different topics, and applications.

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