The Western Blot, also known as immunoblotting, is a widely used analytical technique in molecular biology and immunogenetics. It detects specific proteins within complex samples like tissue homogenates or cell extracts, allowing researchers to identify, visualize, and quantify particular proteins. This provides valuable insights into their presence and relative abundance in various biological processes, disease states, and medical diagnostics like HIV testing.
The technique relies on three main stages: separating proteins by size, transferring them to a solid support, and then identifying target proteins using antibodies. This multi-step approach makes it possible to isolate a protein of interest from a mixture containing thousands of different proteins, contributing to its broad application.
Preparing Samples for Analysis
Before proteins are separated and detected, they are extracted from their biological source, such as cells or tissues. This initial step, lysis, involves breaking cell membranes to release intracellular proteins into a solution. Various lysis buffers are available, chosen based on the protein’s location and properties, like stronger detergents for membrane-bound proteins.
After lysis, cellular debris like nuclei and insoluble components are removed, typically by centrifugation. This pellets heavier debris, leaving solubilized proteins in the supernatant. To prevent protein degradation, samples are often kept on ice or at 4°C, and protease and phosphatase inhibitors are added.
Following extraction, protein concentration in each sample is accurately determined. This ensures equal amounts of total protein are loaded into each gel lane for meaningful comparisons. The Bradford assay is a common quantification method, using a colorimetric reaction where a dye binds to proteins, causing a measurable color change.
Separating Proteins by Size
Proteins are separated by molecular weight using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Proteins are denatured by heating them in a sample buffer containing sodium dodecyl sulfate (SDS) and a reducing agent. SDS coats the proteins, giving them a uniform negative charge proportional to their size.
This uniform negative charge ensures proteins migrate towards the positively charged electrode when an electric current is applied. The polyacrylamide gel acts as a molecular sieve, allowing smaller proteins to move more quickly through its pores than larger ones. The gel consists of a stacking gel that concentrates proteins and a resolving gel that separates them by size.
The gel electrophoresis apparatus holds the gel between two buffer reservoirs, connected to a power supply. Proteins travel through the gel, and their migration distance is inversely proportional to their molecular weight. A protein ladder, with known molecular weights, is run alongside samples to estimate protein sizes.
Transferring Proteins to a Membrane
After proteins are separated by size within the polyacrylamide gel, they are transferred onto a stable solid support membrane. This transfer is critical because the gel is fragile and unsuitable for subsequent antibody incubations. Electrophoretic transfer, or electroblotting, is the most common method.
During electrophoretic transfer, the gel and a membrane (commonly nitrocellulose or PVDF) are placed in a “sandwich” with filter papers and sponges. This assembly is submerged in a transfer buffer within an apparatus. An electric field drives negatively charged proteins from the gel onto the membrane, where they bind.
Proper transfer is important for successful downstream detection. Factors like membrane type, pore size, transfer buffer composition, and electric current duration and strength influence efficiency. PVDF membranes have higher binding capacity for low-abundance proteins, while nitrocellulose is often used for proteins of low to mid-range molecular weights.
Identifying Target Proteins with Antibodies
With proteins immobilized on the membrane, the next stage is identifying the protein of interest using antibodies. First, the membrane is blocked to prevent non-specific antibody binding. This is done by incubating it with a solution containing non-specific proteins, such as non-fat dry milk or bovine serum albumin (BSA).
Following blocking, the membrane is incubated with a primary antibody. This antibody is designed to recognize and bind to a unique site, called an epitope, on the target protein. After incubation, unbound primary antibodies are washed away to reduce background signal.
Next, a secondary antibody is introduced. This antibody does not bind directly to the target protein but binds specifically to the primary antibody. Secondary antibodies are conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). This enzyme component is essential for the final detection step.
For detection, a substrate solution is added that reacts with the enzyme conjugated to the secondary antibody. For HRP, a common detection method is chemiluminescence, where the substrate reacts with HRP to produce light. This light emission is then captured by a digital imager or X-ray film, generating a visible signal that indicates the presence and location of the target protein on the membrane.
Analyzing and Interpreting Results
After detection, the Western Blot membrane displays bands corresponding to the target protein. Analyzing these bands provides information about the protein’s presence, approximate molecular size, and relative abundance. The position of a band relative to molecular weight markers confirms the protein’s size.
The intensity or darkness of a band is proportional to the amount of target protein present. Researchers quantify this intensity using image analysis software to compare protein levels across different experimental conditions. To ensure accurate comparisons, equal protein loading in each lane is verified by using a loading control.
A loading control detects a housekeeping protein, expressed consistently across all samples, to normalize the signal. This helps account for variations in sample loading or transfer efficiency. The absence of an expected band, or appearance of bands at unexpected sizes, can indicate issues like lack of protein, antibody specificity problems, or protein modifications.