译文（可往下滚动看完整翻译）：

1. 00:00 - 00:49实验前准备

I’m Sally Mulford, a senior molecular biologist here in Li-Cor, and I’m gonna take you through the steps to use our Revert™ total protein stain for normalization of two-colour Western Blot. First, prepare your sample and run them on an SDS-PAGE gel as you normally would. Then transfer to a nitrocellulose or PVDF using standard method. Then, for best result, you should completely dry your membrane, and then rehydrate in PBS or TBS. If you have PVDF membranes, then you should briefly rehydrate them in methanol, before putting them in PBS or TBS buffer.

我叫Sally Mulford, LI-COR的高级生物学家。今天我将给大家一步一步演示如何用我们的Revert™ 700总蛋白染料来进行双色Western Blot的均一化实验。首先，将你的常规方法把蛋白样品跑SDS-PAGE胶，并转膜到NC膜或PVDF膜上。转膜后，为了获得更好结果，把膜彻底晾干，然后用PBS或者TBS重新湿润。如果你使用的是PVDF膜，应先用甲醇短暂浸泡湿润后再放入PBS或者TBS缓冲液中。

2. 00:50-01:29染色前准备

For Revert™ staining, make sure that methanol has been added to each bottle and indicated that on each bottle. And once your membrane has been rehydrated in TBS or PBS, pour off the buffer, and then we’ll wash it one time with water, with distilled water.

在使用Revert™ 700总蛋白染料之前，请确保已往里面加入甲醇并作好标记。先把前面用于湿润膜的PBS和TBS倒掉，然后用蒸馏水洗一次。

3. 01:30-02:41染色

Next we’ll add 5 ml of the Revert™ total protein stain. And let it incubate with shaking for 5 minutes. After the 5 minutes, pour off the stain, and then wash briefly two times with the REVERT wash solution. It’s about 5 ml per wash. And then you’re ready to image the membrane on the Odyssey.

下一步，加入5毫升Revert™ 700总蛋白染料。避光震荡（可置于摇床上）孵育5分钟，然后倒掉染料。用5毫升的Revert™清洗膜，洗两次，然后就可以准备用Odyssey上机成像了。

4. 02:42-03:01成像

Place the blue protein stain side down on a clean Odyssey scan bed, and then image on the auto setting. The 700 image is your total protein image to be used for analysis later.

把有蓝色染料的那一面朝下放在Odyssey扫描器上，然后用自动模式参数进行成像。700通道获得的图像就是后续用于分析的总蛋白图像。

5. 03:02-04:22脱色

For the two-colour Western Blot, you wanna add 5 ml of the reversal (destaining) solution to the membrane, and then let it incubate on the shaker for 5 minutes, but don’t exceed 10 minutes. Once the 5 minutes are up, decant the reversal (destaining) solution, rinse briefly with water, then continue to the blocking step of your standard two-colour Western Blot and use, this time, 700 and 800 (infrared dye) conjugated secondary antibodies.

如果后续是要进行双色Western Blot成像，可以先用5毫升的脱色液（Revert Destaining Solution）进行脱色，在摇床上震荡孵育5分钟，但不要超过10分钟。孵育后，倒掉脱色液，用蒸馏水冲洗，然后按照常规的双色Western Blot实验步骤进行封闭，一抗孵育，再到700和800近红外染料抗体孵育。

6. 04:23-04:57双色Western Blot检测

The image to the right is your two-colour image, don’t worry about the residual 700 stain in the lanes, the Image Studio software will substract the background in the analysis phase. The image on the left is the 700-channel total protein image acquired earlier. The  Revert™ software analysis video presented by Dr. Holt will show you how to quantify your target bands and perform the normalization calculation using Image Studio software.

右边图像就是您的双色Western Blot图像，不用担心用到上面700通道的残留染料，Image Studio在分析的时候会把对应背景减掉。左面的700通道图像即是该膜在700通道获得的总蛋白图像。