Western blotting, or immunoblotting, is an analytical technique used in cell and molecular biology to identify specific proteins in a complex mixture of proteins, such as those present in cell or tissue extracts. The technique uses three steps to achieve this: separation by size, transfer to a solid medium and, finally, visualisation. by protein tagging with the use of appropriate primary or secondary antibodies.

Western blotting is a laboratory technique used to detect a specific protein in a blood or tissue sample. The method involves the use of gel electrophoresis to separate proteins from the sample. The separated proteins are transferred from the gel to the surface of a membrane. The membrane is exposed to a specific antibody against the protein under study. The binding of the antibody is detected using a radioactive or chemical marker. A Western Blot is sometimes used to diagnose disease.

Western blotting (also called immunoblotting) is a technique used for the analysis of individual proteins in a mixture of proteins (e.g. a cell lysate).

In Western blotting (immunoblot) the protein mixture is applied to a gel electrophoresis on a carrier matrix (SDS-PAGE, native PAGE, isoelectric focusing, 2D gel electrophoresis, etc.) to sort the proteins by size, charge or other differences in the 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 in which they have been separated due to charge interactions.

The proteins in this immunoblot are then accessible for antibody binding for detection.

Antibodies are used to detect target proteins in the western blot (immunoblot). The antibodies are conjugated to fluorescent or radioactive labels or enzymes that give rise to a subsequent reaction with an applied reagent, resulting in a staining or emission of light that allows detection.

The term Western Blotting is based on a play on words. Southern blotting, which is a method for detecting specific DNA sequences, is named after Ed Southern, who was the first to describe this procedure..

The western blot (immunoblot), as well as the northern blot (for RNA detection), play on the meaning of this name.

The gel electrophoresis is a technique used to separate fragments of DNA (or other macromolecules, such as RNA and proteins) by size and charge. Electrophoresis involves applying a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will move through the gel in different directions or at different speeds, thus separating them from each other.

• Gel electrophoresis is a technique used to separate DNA fragments according to their size.
• DNA samples are loaded into wells (slots) at one end of a gel and an electric current is applied to draw them through the gel.
• The fragments are negatively charged, so they move towards the positive electrode. Since all DNA fragments have the same amount of charge per mass, smaller fragments pass through the gel faster than larger ones.
• When a gel is stained with a pigment that binds to DNA, DNA fragments can be seen as bands, which represent a group of DNA fragments of the same size.

Different types of gel electrophoresis for proteins can be chosen depending on the criteria by which the proteins are to be separated. Some commonly used electrophoretic methods are: SDS-PAGE, native-PAGE and isoelectric focusing.

This is a denaturing method, as it treats the proteins with an anionic detergent SDS (sodium dodilsulphate). The secondary and tertiary structure is destroyed by this process. In addition, the SDS binds to the proteins and thus covers their chemical charges, which makes the proteins equally negatively charged..

Therefore, the following separation occurs only by the size of the polypeptide chains in the polyacrylamide gel.

With this method, native, non-folded and non-denatured proteins can be separated. This method allows the separation of proteins that are inaccessible by other methods. An example would be the separation of modified and unmodified proteins of the same type (e.g. phosphorylated versus non-phosphorylated state of a protein). Native PAGE can also be used to confirm biologically relevant conformations, such as di-, tri- or tetrameric forms of proteins (as opposed to SDS-PAGE, which would separate individual, denatured peptide chains). This method can also detect different complexes of different proteins.

Separation by “Native PAGE” depends on a number of parameters such as 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 protein loads.

This method is based on the fact that a protein has a specific charge at certain pH values. Depending on the pH, acidic and basic functional groups contribute to increase or decrease the total charge of the protein. The isoelectric point is defined as the point at which the total charge of the molecule is zero, because there is an equal amount of negative and positive charges on 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 electrical charge attached to the gel, the protein moves to the point in the gel where the charge of the gel is equal to that of the protein, and the total charge is equal to zero, i.e. the isoelectric point. Therefore, this method is used to separate proteins by their charges, as well as to determine the isoelectric point of a target protein. Separation occurs by the charge of the protein or by the number of basic and acidic groups contained in the protein.

The above methods for protein gel electrophoresis can also be combined to separate proteins. The choice of methods depends on the specific requirements of the experiment.

After separation of the protein mixture, the polypeptide bands are transferred to a membrane support. For this purpose, the membrane is adhered to the gel and this so-called sandwich is transferred to an electrophoresis chamber. It is possible that a part of the SDS is removed and the protein is partially re-saturated, i.e. it regains its 2D and 3D structure. However, the applied electrical charge causes the proteins to move out of the gel vertically in the direction in which they moved in the gel, onto the membrane. In this way, the protein bands bind to the membrane. The “erased” bands are now available for further processing (e.g. for detection of specific proteins with specific antibodies).

Identification of specific antibodies is possible after separation and blotting of the proteins. Specific antibodies (monoclonal or polyclonal) bind to ‘their’ protein band. Non-specific binding antibodies are removed by washing with buffers containing detergents. In addition, the non-specific binding pockets can be blocked before specific antibodies are added.

Primary antibodies are usually applied first, which are then recognised 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.

The western blot (immunoblot) method has several advantages compared to other immuno-sorbent assays (ISA), such as ELISA.

Western blotting (immunoblotting) extends the idea of ELISA by allowing the separation of the protein mixture by size, charge and/or conformation. The described stripping method allows the detection of several targets, in contrast to ELISA, where only one protein can be detected..

As protein gel electrophoresis separates proteins into bands, the size of the target protein/polypeptide can be determined. It is also possible to (semi-)quantify the protein of interest by running an internal quantity standard in parallel with the samples on the gel.

The protein content of the samples can also be compared (“sample A contains more protein than sample B”)..

A disadvantage of western blot (immunoblot) is that it is time consuming (compared to ELISA) and has a high demand in terms of experimenter expertise.

In addition, it requires optimisation of the experimental conditions (i.e. protein isolation, buffers, type of separation, gel concentration, etc.)..

There are many different types and methods of western blotting (immunoblotting). Therefore, it covers very different topics and applications.

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