Immunoblotting is a process in which antibodies are used to detect proteins, utilizing the antigen-antibody interactions of immunity. Western blot and Dot blot are two immunoblotting methods. This technique can answer a number of different questions depending on the approach - determining if proteins are present in a sample, to detect phosphorylated or active variants of a protein, to quantify a particular protein for gene expression analysis, as the end point of chromatin immunoprecipitation (ChIP), or to determine the relative molecular weight of a protein complex. The binding of the antibody is visualized by chemical reactions.
Protein isolation and sample preparation
The basic procedure starts with a protein sample. Proteins are generally obtained from living cells or frozen tissue (from tissue or cell culture, blood, or tissue collected during necropsy/autopsy/biopsy). Today, many of the basic laboratory procedures are available as kits. A researcher simply buys the kit from one of the many bioreagent suppliers, follows the directions, and voila, they have a sample for analysis. Protein extraction involves buffers that separate the proteins from other cellular components, precipitates the proteins out, and then re-suspends the proteins in solution (i.e. dissolves them). For some immunoblotting procedures, the proteins are then purified, which can involve affinity columns, but the exact procedures and buffers depend on the protein(s) of interest, the starting material containing the protein, and the exact immunoblotting procedure being planned.
When the sample is purified protein and a specific antigen against the protein of interest is available, Dot blot can be useful. Essentially the protein sample is dotted at the center of a grid on nitrocellulose paper. After the membrane dries, the area outside the spread of the sample is blocked to avoid nonspecific antibody binding to the membrane (milk is generally used for blocking in immunoblotting techniques). The membrane is incubated with the antibodies and then visualized based on the secondary antibody used. The signal is compared to a standard.
Western blot is now sometimes simply referred to as immunoblot by bioreagent companies. It is nearly the same procedure as Dot blot except that the proteins in the sample are separated on a gel and then transferred to a membrane (nitrocellulose or PVDF). SDS-PAGE is the standard separation procedure – based on the gradient of the polyacrylamide gel, proteins of various sizes can be separated. This procedure can be done once or twice (2D PAGE) depending on how the experiment needs the proteins separated. Electrical current is used to force the proteins out of the gel onto the membrane, which is blocked and incubated with antibodies for visualization.
Primary and secondary antibodies
The antibodies are the key to immunoblotting techniques. The primary antibody recognizes the antigen on the protein of interest and binds to it. This can be affected by the exact epitope the antibody was raised against, so some would recognize the protein if it was only the C-terminal fragment on the membrane, others may recognize only the phosphorylated form of the protein. Choosing the right antibody is important to the aim of the experiment. The secondary antibody then recognizes the primary antibody and binds. If the primary antibody was raised in mice, the secondary would an anti-mouse antibody. The secondary antibody is what creates the bands on images of immunoblots – it is the molecule that allows visualization via chemical reactions. Common conjugates to secondary antibodies are horseradish peroxidase (HRP) and alkaline phosphatase.