Optimizing Chemiluminescent Western Blots – Keep the Blot Wet

In the last post, I mentioned that high background on your chemiluminescent Western blot could be due to excess, pooled substrate or letting the blot dry out. So how do you keep the blot moist?

Well, you can place a clear, flat plastic covering on the blot to prevent it from drying out. This will keep the substrate in contact with the HRP enzyme and keep the blot moist. However, there are a few things to keep in mind when using a plastic covering or wrap.

  1. Image the plastic covering by itself first to determine if it scatters light (which would cause high background). You may need to try several types of plastic coverings before finding the best one.
  2. Handle the wrap and the blot with care. Wrapping the blot in plastic wrap may cause unwanted background, especially if it’s folded or handled roughly. When using plastic wrap it is important to avoid wrinkles, as they scatter light (and, again, give you high background).
  3. Try to avoid leaving fingerprints from pressing on the blot (we’ll talk about fingerprints in another post).

In the image, you can that there is unwanted background caused by the plastic wrap.
Background due to plastic wrap

An alternative to plastic wrap is to use a clear, flat plastic covering, such as a clear sheet protector (from your local office supply store).

IMPORTANT: Be sure to image the sheet or any plastic you use to see if it fluoresces all by itself! After all, this is a creative use of sheet protectors, but not what the manufacturer had in mind so not what they were QC’d for.

And, remember, go digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!