Immunoassay: Western Blotting: Videos & Practice Problems

Western blotting, or immunoblotting, is a technique combining SDS-PAGE gel electrophoresis and antibody detection to identify specific proteins or antibodies, such as HIV antibodies in patient serum. SDS-PAGE separates proteins by size, which are then transferred to a protein-binding blot. Patient primary antibodies bind target proteins, and labeled secondary antibodies enable visualization. This method is crucial in adaptive immunity diagnostics, confirming infections by detecting antigen–antibody complexes, enhancing understanding of immunology and serological testing.

Immunoassay: Western Blotting

Western blotting, also known as immunoblotting, is a powerful laboratory technique used to detect specific proteins within a complex mixture. This method begins by separating proteins through gel electrophoresis, typically using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). SDS-PAGE is essential because it separates proteins based solely on their molecular size, eliminating the influence of charge or shape. During this process, larger proteins migrate more slowly through the gel matrix, while smaller proteins travel faster, allowing for effective size-based separation.

After electrophoresis, the separated proteins are transferred onto a protein-binding membrane, often referred to as a blot. This transfer preserves the spatial arrangement of proteins as they appeared in the gel, enabling subsequent detection steps. The next phase involves incubating the blot with a patient’s serum containing primary antibodies. If the patient has antibodies specific to the target proteins, such as HIV proteins in diagnostic applications, these primary antibodies will bind to their corresponding antigens on the blot.

Since the protein bands and antibody bindings are invisible to the naked eye, visualization requires the addition of labeled secondary antibodies. These secondary antibodies are designed to bind to the primary antibodies and are often conjugated with fluorescent or enzymatic labels. When activated, these labels produce a detectable signal, revealing the presence and position of the target proteins. This detection confirms the presence of specific antibodies in the patient’s serum, providing a reliable diagnostic tool, such as confirming HIV infection.

Western blotting integrates key concepts of molecular biology and immunology, including antigen-antibody specificity and protein separation techniques. The method’s sensitivity and specificity make it invaluable for both research and clinical diagnostics. Understanding the role of SDS-PAGE in size-based protein separation and the use of labeled antibodies for detection is crucial for mastering this technique.

All of the following immunoassay testing techniques are paired with their correct characteristics except which of these answers?

• A. Fluorescent antibody test – microscopy.
• B. ELISA – radioactive label.
• C. Western blot – gel electrophoresis.
• D. All of the above are matched correctly.

Answer: (B)

Which of the following immunoassay techniques would be the best for screening thousands of specimens for COVID-19?

• A. Fluorescent antibody tests.
• B. Western blotting.
• C. ELISA.
• D. None of the above.

Answer: (C)

Here’s what students ask on this topic:

What is the principle behind Western blotting and how does it work?

Western blotting, also known as immunoblotting, is a laboratory technique used to detect specific proteins in a sample. The process begins with SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), which separates proteins based on their size. Proteins are first denatured and coated with SDS, giving them a uniform negative charge, allowing separation solely by size during electrophoresis. After separation, proteins are transferred onto a blotting membrane, which immobilizes them in the same pattern as the gel. The membrane is then incubated with a patient's serum containing primary antibodies that specifically bind to target proteins (antigens). To visualize these antigen-antibody complexes, labeled secondary antibodies that recognize the primary antibodies are added. These secondary antibodies are often tagged with enzymes or fluorescent markers, enabling detection of the protein bands. This method is widely used for protein identification and disease diagnosis, such as confirming HIV infection by detecting antibodies against HIV proteins.

Why is SDS-PAGE used in Western blotting instead of regular gel electrophoresis?

SDS-PAGE is used in Western blotting because it separates proteins based strictly on their size, which is crucial for accurate identification. SDS (sodium dodecyl sulfate) is a detergent that denatures proteins and coats them with a uniform negative charge, masking their native charge differences. This ensures that during electrophoresis, proteins migrate through the polyacrylamide gel matrix solely according to their molecular weight, with smaller proteins moving faster and larger proteins moving slower. Regular gel electrophoresis separates molecules based on both size and charge, which can complicate interpretation. Using SDS-PAGE provides a consistent and reliable way to separate proteins, making it easier to identify specific proteins when they are later detected by antibodies in the Western blotting process.

How do labeled secondary antibodies help in the detection of proteins in Western blotting?

Labeled secondary antibodies play a critical role in the visualization step of Western blotting. After the primary antibodies from the patient's serum bind to the target proteins on the blot, the proteins themselves remain invisible to the naked eye. Secondary antibodies are designed to specifically bind to these primary antibodies. They are conjugated with detectable labels such as enzymes (e.g., horseradish peroxidase) or fluorescent dyes. When the labeled secondary antibodies bind to the primary antibodies, they enable visualization of the protein bands through colorimetric, chemiluminescent, or fluorescent detection methods. This amplification step increases sensitivity and allows researchers to see the presence and approximate quantity of the target proteins, confirming antigen-antibody interactions essential for diagnostics and research.

What are the main steps involved in performing a Western blot?

The main steps of Western blotting include: (1) Protein separation by SDS-PAGE, where proteins are denatured and separated based on size; (2) Transfer of the separated proteins from the gel onto a blotting membrane, which immobilizes the proteins in the same pattern; (3) Incubation of the membrane with primary antibodies from a patient’s serum or experimental sample, which bind specifically to target proteins; (4) Addition of labeled secondary antibodies that bind to the primary antibodies, enabling detection; and (5) Visualization of the protein bands using appropriate detection methods such as chemiluminescence or fluorescence. Each step is essential for ensuring specific, sensitive detection of proteins, making Western blotting a powerful tool in molecular biology and diagnostics.

How is Western blotting used in diagnosing HIV infection?

Western blotting is used in HIV diagnosis by detecting antibodies against HIV proteins in a patient's serum. Known HIV proteins are first separated by SDS-PAGE and transferred onto a blotting membrane. The patient's serum, containing primary antibodies, is then applied to the membrane. If the patient has been exposed to HIV, their antibodies will bind to specific HIV proteins on the blot. Labeled secondary antibodies are added to bind these primary antibodies, allowing visualization of the protein bands. The presence of these bands confirms that the patient has antibodies against HIV, indicating infection. This method is highly specific and was traditionally used as a confirmatory test for HIV after initial screening tests.