Western blot

Summary

Western blot is a molecular biology technique for detecting specific proteins in a sample. It involves denaturing proteins, separating them via SDS-PAGE, and transferring them onto a PVDF membrane. Two transfer methods are described: capillary action using buffer solution and electro blotting with an electric field. After blocking the membrane to prevent non-specific binding, a primary antibody binds the target protein, followed by a secondary antibody with a reporter enzyme that produces a detectable color.

Takeaways

• 🔬 **Western Blot Technique**: Western blot is a molecular biology technique used to detect specific proteins in a sample.
• 🔥 **Protein Denaturation**: Proteins are denatured by heating in boiling water to prepare them for separation.
• 🧬 **SDS-PAGE Separation**: Denatured proteins are separated by size through SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis).
• 📄 **Transfer to PVDF Membrane**: Proteins are transferred to a PVDF (Polyvinylidene Fluoride) membrane, which can be done by capillary action or electro blotting.
• 💧 **Capillary Transfer**: One transfer method involves placing the membrane on the gel and using buffer solution's capillary action to move proteins onto it.
• ⚡ **Electro Blotting**: The alternative transfer method uses an electric field to move proteins towards the positively charged electrodes onto the membrane.
• 🛡 **Blocking Non-Specific Binding**: The membrane is blocked with BSA or milk protein and detergent to prevent non-specific antibody binding.
• 🏹 **Primary Antibody Binding**: A primary antibody specific to the protein of interest is applied to bind to the protein on the membrane.
• 🔍 **Secondary Antibody Detection**: A secondary antibody with a reporter enzyme is used to detect the binding of the primary antibody.
• 🎨 **Color Development**: The reporter enzyme converts a substrate into a colored product, indicating the presence of the desired protein.

Q & A

What is Western blot?

Western blot is a molecular biology technique used to detect specific proteins in a given sample.

How are proteins denatured in Western blot?

Proteins are denatured by heating them in boiling water bath for a few minutes.

What is the purpose of SDS-PAGE in Western blot?

SDS-PAGE is used to separate the proteins present in the sample based on their size.

What is a PVDF membrane and how is it used in Western blot?

PVDF membrane is a special membrane used to transfer proteins from the gel after separation. It is placed on the gel and proteins are transferred onto it either by capillary action or electro blotting.

How does capillary action facilitate protein transfer in Western blot?

Capillary action causes the buffer solution to move upwards, which in turn moves the proteins upwards, attaching them to the PVDF membrane.

What is electro blotting and how does it differ from capillary transfer?

Electro blotting involves applying an electric field to move proteins towards positively charged electrodes, transferring them onto the membrane. It differs from capillary transfer by using an electric field instead of buffer movement.

Why is it necessary to block the membrane before applying the primary antibody?

Blocking the membrane with BSA or milk protein and mild detergents prevents non-specific binding of the antibody to the membrane, ensuring that it binds only to the desired protein.

What is the role of the primary antibody in Western blot?

The primary antibody specifically binds to the desired protein on the membrane, marking its presence for detection.

How does the secondary antibody contribute to protein detection in Western blot?

The secondary antibody, which is attached to a reporter enzyme, binds to the primary antibody. It converts a substrate into a colored product, indicating the presence and location of the primary antibody and, by extension, the target protein.

What is the final step in detecting the desired protein using Western blot?

The final step is to visualize the binding of the secondary antibody to the primary antibody, which is achieved through the conversion of a substrate into a colored product by the reporter enzyme.