Western blotting is a technique used to detect specific proteins in a sample. It can be used to detect the presence of specific proteins, verify the purity of your protein samples, and assess the expression level of proteins in cells. A Western blot utilizes two antibodies: a primary antibody that directly binds to the protein of interest and detects it; and a secondary antibody (also called an HRP conjugated secondary antibody) that binds directly to the primary antibody and helps detect it.
The primary antibody is the first antibody in a Western blot. It is directly conjugated with a fluorophore or enzyme, and binds to the protein of interest. The primary antibody is connected to an immunoglobulin via hydroxyapatite (HA), which is then captured by Protein A or G in your sample.
The secondary antibody is used to detect the primary antibody on the blot. A variety of conjugates are available, including fluorophores (which emit light when excited by a beam of light) and enzymes that catalyze color development in the presence of certain substrates. The most common type of secondary antibody is an enzyme-conjugated goat anti-mouse IgG.
In a direct Western blot, you will use an enzyme-linked antibody that is specific for the target protein. The enzyme that is linked to this antibody will produce color when it binds to the protein of interest. For example, if you're looking for immunoreactive bands from your protein of interest on an SDS-PAGE gel, they can be visualized by adding either Coomassie blue staining or silver staining reagents after electrophoresis.
To perform a direct Western blot, your primary antibody must be conjugated with a fluorophore or enzyme. The conjugation of these proteins to the antibodies enables their detection via an imaging system such as a fluorescent microscope or an ELISA plate reader.
The primary antibody is used to detect the target protein, while the secondary antibody helps detect it. The secondary antibody can be labeled with a fluorescent dye or an enzyme and detects via indirect means. This secondary detection can be achieved using a variety of methods: either by using two different primary antibodies (one for detecting each band), one for each band; or by using an enzyme-linked detection system such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
The protein samples are separated by size and transferred to a membrane. Once the proteins are bound to the membrane, you can probe it with your primary antibody. The primary antibody binds to one of your protein targets—the one that has been labeled with an enzyme called peroxidase in this case—and generates a signal after exposure to substrates such as diaminobenzidine or 3,3’-diaminobenzidine (DAB). The signal is then detected using a secondary antibody coupled with horseradish peroxidase (HRP), which catalyzes a reaction with hydrogen peroxide solution creating another color change visible under light microscopy.
Electrophoresis is a separation technique that uses an electric current to move charged particles through a gel matrix. During electrophoresis, the DNA or RNA sample is loaded into wells in an agarose or acrylamide gel matrix and placed in buffer at pH 8.2 (TBE). The samples are then subjected to an electric field for a specific amount of time, depending on what you're trying to accomplish with your western blotting experiment.
Another way to transfer your sample is by blotting. Blotting is a method that uses paper or nylon membranes as a transfer medium. The advantage of blotting over electrophoresis is that it's faster and less likely to cause damage to the nucleic acids in the sample.
The proteins are then treated so that they can be visualized under UV light. The proteins are stained with a chemical called Coomassie Blue, which makes the proteins appear blue (and sort of sparkly).
While gel electrophoresis is very useful for separating DNA, RNA, or proteins by size and is used routinely in biology labs around the world, it has several disadvantages. For one thing, it requires a lot of equipment and can be difficult to interpret since the resulting bands are sometimes faint. Gel electrophoresis also takes more time than blotting does—it can take hours for a complete gel run while blotting only takes minutes or hours depending on your experimental design.
Gel electrophoresis also relies on radioactive labels that require special handling protocols and disposal procedures that aren’t always practical in day-to-day laboratory workflows. Blotting papers are non-radioactive solutions that don’t require much special equipment beyond what most labs already have: a centrifuge and some reagents (e.g., buffers).
To perform electrophoresis, the nucleic acid sample is loaded into wells in an agarose or acrylamide gel matrix and submerged in an electrophoresis buffer called TBE (Tris-borate-EDTA). The agarose gel contains the nucleic acid sample and the buffer. The EDTA acts as an anticoagulant. The acrylamide gel contains just the buffer because it does not require any proteins to carry out separation of DNA or RNA (RNA is a single-stranded nucleic acid molecule). The samples are loaded into wells for separation based on size and then transferred to a membrane for detection by fluorescently labeled antibodies bound to specific sequences on each strand of DNA or RNA.
The primary antibody is used to identify the protein of interest and usually has a proven specificity for that protein. The secondary antibody binds specifically to domains found on the primary antibody, which are not necessary for its binding ability but can be used to identify or verify the presence of an antigen.
In addition to showing the presence of a particular protein, the secondary antibody can also be used to determine its quantity. This is done by using different concentrations of primary antibodies and comparing their respective fluorescence intensities. However, because each primary antibody has only one binding site, it is not possible to detect multiple proteins with a single Western blot.
To achieve this goal, you'll need two or more sets of primary antibodies that bind different regions on each protein. For example, if you want to detect three different proteins in your sample (A1-A3), then you would need three types of probes: one labelled with biotin that recognizes all three proteins (B1), another labelled with an arbitrary fluorescent dye which recognizes A1 (F2) and finally another labelled with an arbitrary fluorescent dye which recognizes A2-A3 (F3).
The primary antibody is used to identify the protein of interest in a Western blot, so it should be specific for that protein. This means that it will bind only to your target protein and not any other proteins present on the blot. The secondary antibody is used to identify the primary antibody by binding specifically to domains found on the primary antibody's surface.
In summary, Western blots are a powerful technique for identifying the presence or absence of proteins in samples. They depend on two antibodies: one that binds directly to the protein you’re looking for and another that binds to the first one. The second antibody can then be detected by either a fluorescent dye or an enzyme that reacts with it. This results in a band on an agarose gel with your target protein present within it!