Western blotting is an improved methodology used in molecular biology to detect a particular protein in a sample of tissue homogenate or extract. This analytical technique is based on the ability of proteins to bind to specific target antibodies. Primarily, Proteins are separated according to the size by using Gel electrophoresis (SDS PAGE) and then transferred to either nitrocellulose or PVDF membrane.
Sample preparation is the first step in Western Blotting.
1. Pellet the cells by low speed centrifugation.
2. Lyse the cells using a suitable cell disruption method for eg. Sonication to release the protein of interest.
3. Spin down the lysate to pellet the proteins. The cell debris goes to the supernatant.
4. The proteins are then solubilized using Buffers containing detergents.
5. Collect the washed proteins in a fresh tube and proceed with analysis.
The second step in Western blotting is protein separation using Gel electrophoresis.
The proteins in the sample are separated according to the size on the gel. The protein of interest is then transferred to a membrane which is either Nitrocellulose or PVDF using an electric field or by applying pressure. Usually the membranes are sticky and binds all proteins non-specifically.
The binding of protein onto the membrane is based upon hydrophobic interactions as well as charged interactions between the proteins and membrane. The nonspecific binding sites on the membrane are blocked by incubating the membrane in proteinaceous blocking buffer. PVDF membrane needs to be soaked in 100% methanol before use.
The next step is Blocking. The membrane is then blocked, in order to prevent non-specific protein interactions between the antibodies and the membrane. This is usually done by placing the membrane in a solution of BSA or Non-fat milk. Once the membrane is blocked, it is ready to incubate with primary antibody. Incubation is done by diluting the antibody in PBS with some protein (BSA) or 5% skim milk to prevent non-specific binding of the antibody.
The diluted antibody solution and the membrane are incubated in room temperature for 1 hour with gentle agitation. The primary antibody recognizes only the protein of interest and binds to it. The membrane is then rinsed with PBS buffer to remove the unbound primary antibody. Then the secondary antibody is incubated with the membrane. It binds to the primary antibody. This secondary antibody is usually linked to an enzyme that can allow for visual identification of where it has bound on the membrane. The membrane is washed to remove unbound secondary antibodies. Since the primary antibody only recognizes the protein of interest, and the secondary antibody only recognizes the primary antibody, if there is stain present on the membrane, then the protein of interest must also be present on the membrane.
Thus, the protein bands on the membrane that are stained contain the protein that is to be detected. Size estimations can be done by comparing the stained bands to that of a pre-stained protein size marker. If a radioactive label is used, the radioactive membrane can be placed against a medical X-ray film. Bands corresponding to the protein of interest will appear as dark regions on the film.