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!
Okay, it’s football season, and I thought the analogy fit. 🙂 Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.
Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.
One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.
Why?, you ask.
Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.
Nota bene: Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.
On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.
Maximizing Sensitivity on Chemiluminescent Western Blots Technical Note
Go Digital with the Odyssey Fc Chemiluminescent and Infrared Fluorescent Imaging System or the C-DiGit® Chemiluminescent Western Blot Scanner!