The Western blot, or protein immunoblot, is a widely used analytical technique in molecular biology and immunogenetics. It detects, visualizes, and quantifies specific proteins within complex biological samples. This multi-step process combines protein separation by size with antibody-based detection, making it a powerful tool in biological research.
Preparing Samples and Separating Proteins
Sample Preparation and Quantification
The Western blot process begins with preparing biological samples, such as cells or tissues, to extract their protein content. This extraction typically involves lysing the cells, a process that breaks open the cell membrane to release intracellular proteins. Various lysis methods can be employed, including mechanical disruption or the use of detergents.
Following protein extraction, the total protein concentration in each sample is quantified, often using assays like the Bradford or Bicinchoninic Acid (BCA) assay. These colorimetric assays ensure equal amounts of protein are loaded in each lane for accurate quantitative analysis.
SDS-PAGE Separation
After quantification, proteins are separated using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). This technique separates proteins primarily by their molecular weight. Samples are treated with SDS, an anionic detergent that denatures proteins and coats them with a uniform negative charge, masking their intrinsic charge and shape. Reducing agents like beta-mercaptoethanol or dithiothreitol are also added to break disulfide bonds, ensuring proteins unfold into linear chains.
The denatured proteins are then loaded into a polyacrylamide gel, which acts as a molecular sieve. The gel typically consists of a stacking gel and a resolving gel. When an electric current is applied, the negatively charged protein-SDS complexes migrate through the gel matrix towards the positive electrode. Smaller proteins navigate the gel’s pores more easily, migrating faster and further than larger proteins, resulting in separation by size.
Transferring Proteins to a Membrane
Once proteins are separated by SDS-PAGE, they are transferred from the polyacrylamide gel onto a solid support membrane. This transfer is necessary because the gel is fragile and does not allow for efficient probing with antibodies. Common membrane types include polyvinylidene difluoride (PVDF) and nitrocellulose, both with high protein-binding capacities. PVDF membranes are generally more robust and offer higher binding capacity, while nitrocellulose is suitable for many applications.
The transfer process, often called electroblotting, uses an electric field to move the proteins from the gel to the membrane. The gel and membrane are assembled into a “sandwich” with filter papers and placed in a transfer apparatus. An electric current is then applied, causing the negatively charged proteins to migrate out of the gel and bind to the membrane. Two primary methods exist: wet transfer, where the sandwich is submerged in buffer, and semi-dry transfer, which uses less buffer. Wet transfer is often favored for larger proteins and is considered more reliable, while semi-dry transfer is faster.
Detecting Your Target Protein
Blocking
Following protein transfer, the membrane must be treated to prevent non-specific binding of antibodies. This is achieved through blocking, where the membrane is incubated in a solution containing inert proteins, typically 5% non-fat dry milk or Bovine Serum Albumin (BSA). These blocking agents bind to all unoccupied sites on the membrane, ensuring antibodies only bind specifically to the target protein, thereby reducing background signal.
Primary Antibody Incubation
After blocking, the membrane is incubated with a primary antibody. This antibody is specifically designed to recognize and bind to a unique site, or epitope, on the target protein. Following primary antibody incubation, thorough washing steps are performed using wash buffers. These washes remove any unbound primary antibodies, which helps to minimize background noise and increase the signal-to-noise ratio.
Secondary Antibody and Detection
Next, a secondary antibody is added. This antibody does not bind directly to the target protein but instead binds specifically to the primary antibody. Secondary antibodies are chemically linked to an enzyme, such as horseradish peroxidase (HRP), or a fluorophore.
After another series of washes, a substrate is added if an enzyme-conjugated secondary antibody is used. For HRP, a chemiluminescent substrate reacts with the enzyme to produce light, which can then be detected. If a fluorophore-conjugated antibody is used, the fluorophore emits light upon excitation, which is directly detected.
Analyzing Western Blot Results
After signal generation, the Western blot results are captured using imaging systems. Chemiluminescent signals are detected using X-ray film or digital imagers. Fluorescent signals are captured directly by specialized fluorescent imaging systems. The resulting image displays bands on the membrane, with each band representing a detected protein.
Interpreting the results involves identifying the target protein band based on its molecular weight and comparing its position to a protein ladder, which contains proteins of known molecular weights. Quantification of protein levels is performed using densitometry, a process that measures the intensity of the protein bands. This signal intensity is proportional to the amount of target protein present in the sample.
For accurate quantitative comparisons, it is common practice to normalize the signal of the target protein to a loading control. Loading controls, often housekeeping proteins like GAPDH or beta-actin, are proteins expressed consistently across all samples and serve as an internal reference to account for variations in protein loading or transfer efficiency. Proper normalization enables robust relative comparisons of protein expression levels.