So, you’ve selected your primary antibodies with care (see Know Thy Primary Antibody), and you’re using a great HRP-conjugated secondary antibodies. NOW – what about the chemiluminescent substrate?
Yes, I know, there are tons of different substrates and vendors out there – all claiming to be the best, right? So, how do you choose the right one for your chemiluminescence Western blot?
One thing to keep in mind is that in this wide variety of chemiluminescent substrates for HRP detection, there are some that are better suited for digital Western imaging than others. In general, choose a substrate with a faster rate of reaction for use with the Odyssey Fc Dual-Mode Chemiluminescent and IR Fluorescent Imaging System or other digital chemiluminescent imaging systems.
Some substrates that are designed for optimal performance on film may not be suitable for detection on a CCD-based imaging system. Try different substrates to find the one that gives the most desirable image. As you can see from the images below, the substrate you pick DOES make a difference! So choose carefully!
In the three images below, two-fold serial dilutions of HRP-conjugated secondary antibody (1 ng-1.25 fg) were spotted onto nitrocellulose using a slot blot apparatus. Blots were detected with various chemiluminescent substrates.
Here are 2 documents with more troubleshooting information:
Updated February 18, 2015.
Tired of the darkroom being down or the expense of the development chemicals? Oh, AND all that film you go through because you have to do multiple exposures to get the image just right? Go DIGITAL with the Odyssey® Fc Dual-Mode Imaging System.
Dual-mode? Yes! The Odyssey Fc provides the ability to streamline your chemiluminescent Western blot imaging – no film, no darkroom, just clear fast blot images.
PLUS this digital imaging system includes two near-infrared fluorescent channels for sensitive, quantitative infrared Western blot imaging. So use ECL™ or whatever chemiluminescent substrate you usually use but eliminate film (more on best substrates to use in a later post). See how much money you will save going digital!
The Odyssey Fc System can acquire images in both fluorescent and chemiluminescent modes. Learn about FieldBrite™ XT2 technology and the advantages it gives you with the Odyssey Fc.
The Odyssey Fc Imaging System can also be used for Coomassie-stained protein gel imaging and DNA gel imaging of either ethidium bromide-stained gels or SYTO® 60 stained gels.
We’ve discussed some hints on how there can be considerable difference in primary antibodies – so “Know thy Primary Antibody.“
In addition, we’ve received some questions that are frequently asked – hence called frequently asked questions or FAQs – about primary and secondary antibodies when doing chemiluminescent Western blots. So here they are. I am sure there are more. . .so send them our way by commenting on this post!
Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.
Does it matter where I purchased the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. As well, the ratio of HRP enzyme to antibody varies, and may affect the detection of the target. If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.
Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.
What questions do you have?
Optimizing Chemiluminescent Western blots Technical Note might be a good place to start to get some of those questions answered. And remember you save time and money by going digital with the Odyssey® Fc Chemiluminescent and Infrared Fluorescent Imaging System!
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.
- 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.
- 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).
- 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.
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!
You do chemiluminescent Western blots – you do TONS of them. Sometimes, as I am sure you have discovered, you have ‘challenges’ – the darn thing just doesn’t look right!
What are some things that may happen when using chemiluminescence detection for Western blotting and how do you resolve them? The next series of blog posts here will offer up some tips on how to optimize chemiluminescent Westerns. These tips were designed for Westerns detected with a digital imager, like the Odyssey Fc Chemiluminescent and Near-infrared Fluorescent Imaging System. But many will also apply if you are (still) in the darkroom and using film. 🙂
Pools of excess substrate on the membrane can lead to areas of high background, as can adding more substrate to a membrane that has dried. Apply the substrate carefully and wick off any pools of substrate before imaging. Do not allow the membrane to dry.
The image above is an example of high background due to pooled excess substrate. If your blot looks like the image below, then the membrane dried out and then someone tried to add more substrate.
Optimizing Chemiluminescent Blots on the Odyssey Fc Imager