Name: Western Blotting
Uploaded: 2025-06-14T12:40:35.000Z
Duration: 9 min 57 s
Description: Thousands of YouTube videos with English-Chinese subtitles! Now you can learn to understand native speakers, expand your vocabulary, and improve your pronunciation...

In this video, you will learn how to perform a western blot.

Advanced passive voice

A western blot can be used to identify specific proteins in a sample and provide information about the protein size and relative abundance in the sample.

You will be using this buffer to equilibrate your gel prior to starting the western blot.

Next, remove the gel from the gel cassette using the opening key.

Line up the arrows on the opening lever with the four arrows on the cassette to open the cassette.

After trimming the top and bottom of the gel with a straight edge, equilibrate the gel in the tray with blotting buffer for 15 minutes on a rocking platform.

Pre-soak fiber pads in blotting buffer so that they are thoroughly soaked.

To make a blotting sandwich, obtain a container large enough to fit the gel holder and add enough blotting buffer until the container is filled approximately one centimeter deep.

Place the gel holder cassette in the container with the black side down and immersed in the out of the buffer.

Lay one fiber pad flat on the black plastic.

Next, wet a piece of blotting paper and place it on top of the pad.

Be careful to avoid any bubbles between the pad and the paper and make certain the buffer covers the paper.

Take the gel and carefully place it squarely onto the blotting paper, again being careful to avoid any bubbles between the gel and the blotting paper.

Next you will be applying a piece of nitrocellulose membrane.

Remove the protective sheet from the membrane and wet the membrane with blotting buffer.

Carefully place the membrane squarely on the gel.

Avoid moving the membrane once placed on the gel as proteins will begin to blot immediately.

Using a roller, remove any air bubbles between the gel and the membrane.

Place a second sheet of wet blotting paper on top of the nitrocellulose membrane.

Place a second wet fiber pad on top of the blotting paper.

Fold the clear plastic side of the gel holder over the sandwich and clamp it to the black plastic side by sliding over the white clip.

This tight fit will squeeze the sandwich together.

Insert the gel holder into the inner module.

Make certain that the black side of the gel holder is next to the black side of the module.

Place the inner module into the electrophoresis chamber.

Add a frozen cooling unit and fill the chamber with blotting buffer to the level of the white clip on the gel holder.

Place the lid on the electrophoresis chamber.

Connect the electrical leads to the power supply, making sure the connections are correct, red to red and black to black.

Turn on the power supply and run the blot at 20 volts.

If a timer is available, set it for two and a half hours.

When the run is complete, turn off the power supply and disassemble the electrophoresis chamber and remove the inner module.

Open the module and place it in a container filled with blotting buffer with the black side down.

Starting with the first fiber pad, run the blot at 20 volts.

Remove each layer until you reach the nitrocellulose membrane.

As you remove the membrane, note that the proteins have been transferred from the gel to the membrane.

Note that the kaleidoscope pre-stain standards have been transferred and can be seen on both sides of the membrane.

You can also see that there are no longer any proteins on the gel.

Immerse the membrane in 25 milliliters of blocking solution and incubate it for 15 minutes at room temperature on a rocking platform.

Incubate for 10 to 20 minutes on a rocking platform.

The platform should be set at a faster setting to ensure constant coverage of the membrane.

Rinse the membrane quickly in 50 milliliters of wash buffer and then discard the wash buffer.

Add another 50 milliliters of wash buffer to the membrane and let it wash for 3 minutes on the rocking platform at a medium speed setting.

Add 10 milliliters of secondary antibody and incubate the membrane for 5 to 15 minutes on the rocking platform at a fast speed.

Rinse the membrane quickly in 50 milliliters of wash buffer and discard the wash buffer.

Add an additional 50 milliliters of wash buffer and wash the membrane for 3 minutes on the rocking platform on a medium speed setting.

Incubate the membrane with the substrate for 10 to 30 minutes with either manual shaking or on a rocking platform.

Once the colors have developed, rinse the membrane twice with distilled water and blot dry with a paper towel.

Air dry for 3 to 60 minutes and then cover in plastic wrap for storage.