There is no residual electron spin, and this is called the singlet excited state. The 3-aminophthalate is produced with an unpaired electron in a non-ground-state orbital. However, we must understand that one product of the reaction is the dianion 3-aminophthalate. The reaction of luminol with an oxidizing agent is complicated and contains many steps. That’s why the ECL reagent is prepared at pH 8.5, which is slightly basic. For the reaction to proceed, luminol needs to be deprotonated. It emits light when it reacts with an oxidizing agent. Luminol produces the light needed to detect the target protein. Let’s explain this by going through the four ingredients of ECL one by one. We will make our own ECL as two separate solutions, too.Īnd in case you are wondering, we keep the hydrogen peroxide separate from the luminol and p-coumaric acid, and both solutions are buffered with the tris-HCl. That is why ECL is supplied as two separate solutions that we mix immediately before the detection step in our Western blot. These are:Īs mentioned, these chemicals react to produce light. What Are the Components of ECL?Įxcluding water, ECL has four ingredients. And because the secondary antibody binds specifically to a primary antibody that binds specifically to a target protein, the emission of light from ECL is proportionate to the amount of that target protein. In a Western blot, the secondary antibody is conjugated to horseradish peroxidase. This reaction is catalyzed by the enzyme horseradish peroxidase. The chemicals in ECL, when mixed together, react to produce light. As you probably know, we use it to detect and quantify specific proteins on a membrane. What Is ECL?ĮCL stands for Enhanced Chemiluminescence. This will improve your Western blot success rate and put you in control if things go wrong.
#ECL SOLUTION WESTERN BLOT HOW TO#
We’re going to learn how to make ECL and explain what its constituent chemicals do. So why not save money and make a private stash of ECL? It’s expensive, contains unstable and light-sensitive chemicals, and the stock solution gets abused by careless researchers. What’s more disappointing than a failed Western blot?Īfter the transfer step, the second biggest cause of failed Western blots is the ECL reagent.
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