Name: Western 印跡 (Western Blotting)
Uploaded: 2025-06-14T12:40:44.000Z
Duration: 9 min 57 s

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

在本視頻中，您將學習如何進行 Western 印跡。

進階被動語態

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.

Western 印跡可用於鑑定樣本中的特定蛋白質，並提供有關樣本中蛋白質大小和相對丰度的資訊。

首先在托盤中裝入印跡緩衝液。

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

在開始 Western 印跡前，您將使用該緩衝液來平衡凝膠。

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

然後，使用打開鍵將凝膠從凝膠盒中取出。

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

將打開杆上的箭頭與盒體上的四個箭頭對齊，即可打開盒體。

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.

用直尺修整凝膠的頂部和底部，然後在搖臺上用印跡緩衝液平衡盤中的凝膠 15 分鐘。

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

將纖維墊預先浸泡在吸墨緩衝液中，使其完全浸透。

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.

要製作印跡夾層，需要一個足以裝下凝膠支架的容器，並加入足夠的印跡緩衝液，直至容器裝滿約一釐米深。

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

將凝膠固定盒放入容器中，黑色面朝下，浸入緩衝液中。

Lay one fiber pad flat on the black plastic.

將一個纖維墊平放在黑色塑膠上。

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

然後，將一張吸墨紙弄溼，放在化妝棉上。

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

注意避免緩衝墊和紙張之間出現氣泡，並確保緩衝墊覆蓋紙張。

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.

取下凝膠，小心地將其平鋪在吸墨紙上，再次注意避免凝膠和吸墨紙之間產生氣泡。

Next you will be applying a piece of nitrocellulose membrane.

接下來，您將使用一塊硝化纖維膜。

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

從膜上撕下保護膜，用吸墨緩衝液潤溼膜。

Carefully place the membrane squarely on the gel.

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

將膜放在凝膠上後避免移動，因為蛋白質會立即開始吸附。

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

用滾筒去除凝膠和薄膜之間的氣泡。

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

在硝酸纖維素膜上再放一張溼吸墨紙。

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

在吸墨紙上再放一塊溼纖維墊。

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.

將凝膠支架的透明塑膠面折到夾層上，然後滑過白色夾子將其夾到黑色塑膠面上。

This tight fit will squeeze the sandwich together.

這種緊密貼合會將三明治擠在一起。

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.

確保凝膠支架的黑色面緊靠模塊的黑色面。

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.

添加冷凍冷卻裝置，並將吸墨緩衝液注入腔室，使其與凝膠支架上的白色夾子持平。

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.

將電氣導線連接到電源，確保連接正確，紅對紅，黑對黑。

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

打開電源，以 20 伏電壓運行印跡。

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

如果有計時器，可將其設置為兩個半小時。

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

運行完成後，關閉電源，拆卸電泳槽並取出內模塊。

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

打開模塊，將其放入裝有吸墨緩衝液的容器中，黑色面朝下。

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

從第一個纖維墊開始，以 20 伏電壓運行印跡。

Remove each layer until you reach the nitrocellulose membrane.

移除每一層，直至硝化纖維膜。

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

移除膜時，注意蛋白質已從凝膠轉移到膜上。

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

請注意，萬花筒預染色標準已經轉移，可以在膜的兩面看到。

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

您還可以看到凝膠上不再有任何蛋白質。

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

將膜浸入 25 毫升的阻斷液中，室溫下在搖臺上孵育 15 分鐘。

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.

平臺的設置速度應較快，以確保持續覆蓋薄膜。

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

用 50 毫升洗滌緩衝液快速沖洗膜，然後丟棄洗滌緩衝液。

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.

再向膜中加入 50 毫升洗滌緩衝液，然後在搖臺上以中速洗滌 3 分鐘。

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

加入 10 毫升二抗，在搖床上快速孵育膜 5 至 15 分鐘。

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

用 50 毫升洗滌緩衝液快速沖洗膜，並丟棄洗滌緩衝液。

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

再加入 50 毫升洗滌緩衝液，在搖臺上以中速洗膜 3 分鐘。

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

將膜與底物一起孵育 10 至 30 分鐘，可手動搖動或放在搖動平臺上。

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

顯色後，用蒸餾水沖洗膜兩次，然後用紙巾吸乾。

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

晾晒 3 到 60 分鐘，然後用保鮮膜覆蓋保存。

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