Question

Why is lysozome (not BSA) used for the standard Folin-Ciocalteu Assay?

Answer 1

BSA is commonly used as a standard, as it is easy to get, inexpensive, and, most importantly, of very high purity. The critical thing is to determine if the enzyme you plan to use as a standard is all pure protein. Assuming the enzyme is in the form of a dry powder, you need to be sure that when you weigh it out to prepare the standard, all of the dry weight is pure protein, without any salts, sugar, etc. that might also be of the material. e.g., If you weight out 10 mg of dry material but it's only ~70% pure, it will cause quantitation errors when you use it as a standard. If it is impure but you know the % purity, you can still use it, but you will need to apply a correction factor based on the % purity. The manufacturer should be able to provide you with this information in a COA (Certificate of Analysis) of that particular batch of lipase.

BSA is probably your best bet. Other proteins that work well are ovalbumin (egg albumin) and lysozyme. Pure lysozyme is easy to get.

Be aware that it is common for different proteins to have different reactivities with protein detection reagents. In the Lowry assay, the final color is a result of the reagents reacting w/ peptide bonds and aromatic amino acids. All proteins have peptide bonds, but they don't all have the same % of aromatic amino acids, and this can affect the final absorbance value for a given weight of protein in the assay. If you were to compare, e.g., BSA w/ lysozyme, using equal weight aliquots of the 2 proteins, you'd get 2 good standard curves, but they won't coincide because of this phenomenon. This is also a good reason not to switch from one protein standard to another during the course of your research, as it can cause inconsistencies in quantitation.

You might also want to consider using the BCA protein assay method instead of the Lowry (BCA = Bicinchoninic Acid). It has an alkaline carbonate reagent similar to that in the Lowry but uses BCA instead of the Folin Ciocalteu reagent of the Lowry. It's as easy to perform as the Lowry and more sensitive.