Nothing is more frustrating in the lab than trying to troubleshoot a multi-step protocol. What do you do when you get to the end of your chemiluminescent Western and you have little to no signal?
You systematically troubleshoot the protocol by using the appropriate controls:
- Samples – good
- Gel – good
- Transfer – good
- Primary antibody – good
- Secondary antibody – good
- Substrate – Not good – What?!
Substrate is probably the last reagent you would suspect to be giving you problems in your chemiluminescent Western. It is quality controlled by the manufacturer, well within the expiration period and you stored it properly. Did the company send you a bad batch?
But there are several things you could be doing that could decrease the likelihood that your substrate is working.
Altering the pH
Whether you realize it or not, if you mix your wash buffer with the substrate, you could be changing the pH of the substrate solution, decreasing it’s ability to work.
Chemiluminescent substrates, like Quantum and Sirius, are made to work at pH 9. Wash buffers are generally in the pH 7 range. If you do not completely drain the wash buffer off of your membrane or do not add sufficient substrate solution to your membrane, you will lower the pH of the substrate.
The solution is simple: make sure you drain off your wash buffer. You can use adsorbent paper to help you. I drop my membrane onto filter paper briefly to wick off moisture. Others hold a lab tissue to the corner of the membrane as they suspend it with tweezers.
In addition, always, always use enough substrate to cover your membrane. Advansta recommends using 1 mL/cm 2.
Drying the membrane after substrate exposure
Membranes should not be allowed to dry out after being incubated with substrate and while exposing to film or being digitally imaged. A dry membrane will not perform correctly.
Make sure you use enough substrate during incubation (1 mL/cm 2) and wrap your blot in plastic wrap or use plastic folders to keep the membrane damp.
Use the right substrate for the experiment
Not all substrates are created equal. In fact, substrates with different sensitivities have been developed to help you detect abundantly-expressed proteins as well as low abundance proteins.
If you try to detect a large amount of protein with a substrate designed to detect small amounts of protein, such as Sirius, you will have problems. This is because you can have too much of a good thing. The excess HRP enzyme bound to the secondary antibody becomes saturated with signal and will eventually die out, giving you variable performance.
Likewise, if your protein is not very abundant, you need to use the extra sensitive substrate.
To avoid this, make sure you pair your substrate with your experiment.
Want to always know your substrate is working?
Wouldn’t it be neat to automatically have a way of checking whether your substrate is working? Advansta has you covered. The WesternBright ChemiPen for marking your membranes contains a substance that reacts with HRP substrates.
Never be in the dark again!
Photo courtesy of Cory Doctorow