Immunoblot

Immunoblot

The immunoblot, also known as Western blot, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample. This method involves the separation of proteins by gel electrophoresis, transfer to a membrane, and detection using antibodies. The immunoblot is a powerful tool for the identification and characterization of proteins, and it is commonly used in research and clinical diagnostics.

History

The technique was first described by Harry Towbin and colleagues in 1979. It was named "Western blot" by W. Neal Burnette in 1981, as a play on the Southern blot, a method for DNA detection developed by Edwin Southern.

Principle

The immunoblot technique involves several key steps:

1. Sample Preparation

Proteins are extracted from cells or tissues using a lysis buffer. The sample is then quantified to ensure equal loading of protein across all lanes of the gel.

2. Gel Electrophoresis

Proteins are separated based on their size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins are denatured and coated with SDS, which imparts a negative charge, allowing them to migrate towards the anode when an electric field is applied.

3. Transfer to Membrane

The separated proteins are transferred from the gel to a membrane, typically made of nitrocellulose or polyvinylidene difluoride (PVDF). This step is crucial for the subsequent detection of proteins.

4. Blocking

The membrane is incubated with a blocking solution, usually containing non-fat dry milk or bovine serum albumin (BSA), to prevent non-specific binding of antibodies.

5. Antibody Incubation

The membrane is incubated with a primary antibody specific to the target protein. After washing, a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP), is applied. This secondary antibody binds to the primary antibody.

6. Detection

The enzyme on the secondary antibody catalyzes a reaction with a substrate to produce a detectable signal, often chemiluminescent or colorimetric. The signal is captured on film or a digital imaging system.

Applications

Immunoblotting is used in various applications, including:

• Protein Identification: Confirming the presence of a specific protein in a sample.
• Protein Quantification: Estimating the relative amount of a protein.
• Post-translational Modifications: Detecting modifications such as phosphorylation or glycosylation.
• Clinical Diagnostics: Diagnosing diseases such as HIV and Lyme disease.

Advantages and Limitations

Advantages

• High specificity due to the use of antibodies.
• Ability to detect proteins in complex mixtures.
• Versatility in detecting post-translational modifications.

Limitations

• Time-consuming and labor-intensive.
• Requires high-quality antibodies.
• Quantification can be challenging due to variability in antibody binding.

Also see

• Southern blot
• Northern blot
• ELISA
• SDS-PAGE
• Antibody