Western Blotting

Summary

This video provides a step-by-step guide to performing a Western blot, a laboratory technique used to identify specific proteins in a sample. The process involves preparing a blotting buffer, transferring proteins from a gel to a nitrocellulose membrane, and incubating with primary and secondary antibodies. After several washes, a substrate is added to visualize the proteins through color development. The video covers each stage in detail, from gel preparation to membrane processing, ensuring clear instructions for optimal results in protein analysis.

Takeaways

• 😀 Prepare the blotting buffer and soak the gel in it for 15 minutes on a rocking platform before starting the Western blot.
• 😀 Carefully remove the gel from the cassette using the opening key and trim the top and bottom of the gel.
• 😀 Create a blotting sandwich by layering soaked fiber pads, blotting paper, gel, nitrocellulose membrane, and another layer of blotting paper and fiber pads.
• 😀 Ensure that the blotting sandwich is tightly clamped together to prevent movement and air bubbles.
• 😀 Set up the electrophoresis chamber with blotting buffer, cooling unit, and connect the power supply to run at 20V for 2.5 hours.
• 😀 After electrophoresis, disassemble the chamber, remove the gel holder, and expose the membrane to reveal transferred proteins.
• 😀 Immerse the membrane in blocking solution for 15 minutes at room temperature to block non-specific binding sites.
• 😀 Incubate the membrane with primary antibody for 10-20 minutes, ensuring constant rocking to cover the membrane evenly.
• 😀 Wash the membrane with wash buffer between antibody incubations to remove excess antibody and minimize background.
• 😀 Incubate the membrane with secondary antibody for 5-15 minutes, then wash again with wash buffer before proceeding to substrate detection.
• 😀 Add substrate and incubate the membrane for 10-30 minutes, monitoring for color development, then rinse and blot dry before storing the membrane.

Q & A

What is the purpose of a Western blot?

A Western blot is used to identify specific proteins in a sample, providing information about the protein's size and relative abundance.

Why is blotting buffer used in the Western blot procedure?

Blotting buffer is used to equilibrate the gel prior to starting the Western blot, ensuring that the gel is properly prepared for protein transfer.

How should the gel be handled when removing it from the cassette?

To remove the gel from the cassette, use the opening key to align the arrows on the lever with those on the cassette, then carefully open it and trim the top and bottom of the gel.

What is the purpose of the blotting sandwich?

The blotting sandwich is created to transfer proteins from the gel to the nitrocellulose membrane. It consists of fiber pads, blotting paper, the gel, and the membrane, ensuring a proper transfer under electrical conditions.

What should be done to the nitrocellulose membrane before placing it on the gel?

Before placing the nitrocellulose membrane on the gel, remove its protective sheet and wet it with blotting buffer to ensure proper interaction during the transfer.

How should the membrane be positioned to avoid issues during the protein transfer?

The membrane should be carefully placed on the gel without moving it once positioned, as proteins will begin to transfer immediately. Avoid air bubbles between the gel and the membrane to ensure efficient transfer.

What is the role of the electrophoresis chamber in the Western blot procedure?

The electrophoresis chamber is used to apply an electric current that facilitates the transfer of proteins from the gel to the nitrocellulose membrane.

Why is the power supply set to 20 volts during the Western blot process?

The power supply is set to 20 volts to drive the proteins from the gel onto the nitrocellulose membrane. This low voltage helps ensure an effective but gentle transfer process over 2.5 hours.

What should be done after the protein transfer is complete?

After the transfer is complete, the power supply should be turned off, and the electrophoresis chamber disassembled. The membrane should be removed from the gel holder and handled carefully to observe the transferred proteins.

What is the purpose of blocking the membrane before antibody incubation?

Blocking the membrane with a blocking solution prevents non-specific binding of antibodies to the membrane, ensuring that the antibodies will only bind to the target proteins.

What is the role of the primary and secondary antibodies in the Western blot?

The primary antibody binds specifically to the target protein, while the secondary antibody binds to the primary antibody and is used to amplify the signal, making it easier to detect the protein of interest.

What is the purpose of using a substrate in the Western blot process?

The substrate is used to react with the secondary antibody, producing a color change that allows the protein bands to be visualized on the membrane.

How should the membrane be handled after color development?

After color development, the membrane should be rinsed with distilled water, blotted dry with a paper towel, and allowed to air dry for 3 to 60 minutes before being wrapped in plastic for storage.