Western blot protocol video

Summary

This video provides a detailed guide to Western blotting, a technique used to separate and detect proteins. It covers sample preparation, gel electrophoresis, protein transfer to a membrane, blocking, antibody incubation, and detection. Key steps include heating samples to denature proteins, running a gel to separate proteins by size, transferring them to a membrane, and using antibodies for protein detection. The process also highlights the importance of optimizing antibody concentration and incubation times. Detection methods vary, from enzyme-linked substrates to fluorescent imaging, allowing researchers to visualize protein bands for analysis.

Takeaways

• 😀 Western blotting separates proteins by molecular weight on a gel, and detects them using antibodies.
• 😀 Protein samples are heated in a sample buffer with a reducing agent, like beta mercaptoethanol, to linearize proteins.
• 😀 Acrylamide gel percentage is adjusted based on the molecular weight of the target protein.
• 😀 Equal amounts of protein should be loaded into each well for accurate results.
• 😀 The gel is run until the dye front moves sufficiently down the gel.
• 😀 Proteins are transferred from the gel to a membrane, typically nitrocellulose or PVDF.
• 😀 The transfer stack includes a gel, membrane, filter paper, and sponges, arranged between the positive and negative electrodes.
• 😀 After protein transfer, the membrane can be stained with Ponceau S solution to confirm successful transfer.
• 😀 The membrane is blocked with a solution like milk or BSA to prevent non-specific antibody binding.
• 😀 Primary antibodies are incubated with the membrane, followed by secondary antibody incubation for detection.
• 😀 Detection systems vary and include enzyme-conjugated secondary antibodies or fluorescent antibodies, with imaging done via X-ray or digital systems.

Q & A

What is the purpose of western blotting?

The purpose of western blotting is to separate proteins on a gel according to their molecular weight, and then transfer them onto a membrane where they can be detected using antibodies.

Why is the sample heated to 95°C in a sample buffer containing a reducing agent?

Heating the sample at 95°C in a sample buffer with a reducing agent like beta mercaptoethanol linearizes the proteins and imparts a negative charge to them, which is proportional to their size.

What is the role of the molecular weight marker in western blotting?

The molecular weight marker is loaded into the first lane of the gel to help determine the size of the proteins in the sample by comparing their migration patterns to the known marker.

How do you ensure the gel is prepared properly for electrophoresis?

To ensure proper gel preparation, make sure the wells are covered with buffer, load equal amounts of protein into the wells, and use the recommended voltage as per the gel and tank manufacturer.

What materials are commonly used as membranes in western blotting?

Membranes for western blotting are commonly made from nitrocellulose or polyvinylidene fluoride (PVDF).

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

Blocking the membrane prevents non-specific binding of antibodies to the membrane by covering potential binding sites with a blocking agent, such as 5% milk or bovine serum albumin (BSA).

How do you prepare the membrane for antibody detection?

After blocking the membrane, dilute the primary antibody in the same buffer and incubate it on the membrane. After primary antibody incubation, wash the membrane thoroughly before applying the secondary antibody.

What is the typical washing procedure after incubating the membrane with the primary antibody?

The membrane should be rinsed twice with wash buffer, followed by a 15-minute wash and three 10-minute washes on a rocker to remove unbound antibodies.

How does the detection step vary depending on the secondary antibody?

If the secondary antibody is conjugated to an enzyme, you need to incubate the membrane with an appropriate substrate before imaging. If the secondary antibody is conjugated to a fluorescent marker, you can proceed directly to imaging without further steps.

What are some imaging options available for detecting protein bands?

Protein bands can be detected using X-ray film or a digital imaging system. Exposure times will need to be optimized to clearly detect the bands corresponding to the proteins of interest.