Western blotting technique | principle and step by step procedure

Summary

This video tutorial delves into Western blotting, a technique for detecting proteins in a mixture. It covers the process from protein extraction to separation via SDS-PAGE, transfer to a membrane, and detection using specific antibodies. The method's sensitivity allows for the detection of nanograms of protein and is crucial for understanding cellular processes and protein interactions. The tutorial also explains the importance of blocking to reduce background noise and the use of primary and secondary antibodies for accurate protein identification.

Takeaways

• 🧬 Western blotting is a technique used to detect proteins in a mixture, different from Southern and Northern blotting which detect DNA and RNA respectively.
• 🔍 It is highly sensitive, capable of detecting nanograms of proteins even when mixed with other proteins.
• 🧴 The process begins with protein extraction from cells using detergent to break down the cell membrane, followed by centrifugation to separate the soluble protein components.
• 🔬 Separation of proteins is achieved through SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which separates proteins based on their mass and charge.
• 🚫 The gel is fragile, so proteins are transferred onto a more robust medium like nitrocellulose or nylon membrane for further analysis.
• 🖨 The transfer of proteins from the gel to the membrane is facilitated by applying an electric current, a process known as electroblotting.
• 🚫 To prevent non-specific binding of antibodies, a blocking step is introduced where the membrane is treated with agents like dry milk to cover non-target areas.
• 🔗 The primary antibody specifically binds to the target protein, but does not provide a signal for detection.
• 🔄 After the primary antibody binding, unbound antibodies are washed away, and a secondary antibody is applied which acts as a reporter.
• 💡 The secondary antibody is linked to an enzyme, often horseradish peroxidase (HRP), which upon reaction with a substrate, produces a detectable signal, such as light or color.
• 📝 The final signal, whether luminescent or colored, indicates the presence and location of the target protein on the membrane, allowing for its identification and analysis.

Q & A

What is Western blotting?

Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by size using SDS-PAGE and then transferring them onto a membrane to be detected with specific antibodies.

How does Western blotting differ from Southern and Northern blotting?

While Southern blotting is used to detect DNA and Northern blotting for RNA, Western blotting is specifically used for detecting proteins. Southern and Northern blotting techniques are quite similar, but Western blotting involves different steps, including the use of antibodies for detection.

What is the sensitivity of Western blotting?

Western blotting is a highly sensitive technique capable of detecting nanograms of protein molecules even in a mixture of other proteins.

What is the first step in the process of Western blotting?

The first step in Western blotting is the extraction of proteins from the cell. This involves treating the cell with a detergent to break down the cell membrane, followed by centrifugation to separate the soluble protein components from cellular debris.

What is the purpose of SDS in Western blotting?

SDS, or sodium dodecyl sulfate, is used in the electrophoresis step of Western blotting to denature proteins and give them a uniform negative charge, allowing them to be separated based on their mass during SDS-PAGE.

What is the role of the polyacrylamide gel in SDS-PAGE?

The polyacrylamide gel in SDS-PAGE provides a matrix with different pores that allow proteins to be separated based on their size. Smaller proteins can migrate further through the gel, while larger proteins are hindered, resulting in bands of proteins based on their mass.

Why is it necessary to transfer proteins from the gel to a membrane in Western blotting?

Transferring proteins to a membrane is necessary because the membrane is more robust and easier to handle than the gel. It allows for the application of specific antibodies for detection and is less prone to damage during the probing process.

What is the purpose of the blocking step in Western blotting?

The blocking step is used to prevent non-specific binding of antibodies to the membrane. It involves applying a blocking agent, such as dry milk, to cover areas of the membrane where the target protein is not present, reducing background noise in the final detection.

What are primary and secondary antibodies in the context of Western blotting?

Primary antibodies are specific to the target protein and bind directly to it. Secondary antibodies, which are anti-primary antibodies, bind to the primary antibodies and are often linked to an enzyme or a reporter molecule that aids in the detection of the target protein, such as through a colorimetric or luminescent reaction.

How is the presence of the target protein detected after the application of primary and secondary antibodies?

After the application of primary and secondary antibodies, a substrate is added that reacts with the enzyme linked to the secondary antibody. This reaction produces a visible color change or light emission at the location of the target protein, indicating its presence on the membrane.

What is the significance of the enzyme HRP in Western blotting?

HRP, or horseradish peroxidase, is an enzyme commonly linked to the secondary antibody in Western blotting. It catalyzes the conversion of a substrate, such as luminol, into a product that emits light, allowing for the detection of the target protein on the membrane.