The casein assay detects the peptides released from casein by the action of protease. The protein assay will also detect the peptidic inhibitors, potentially causing an over-measurement if the concentrations of inhibitors are high enough. One approach to deal with this problem would be to include a blank sample for each concentration of each protease inhibitor, omitting the casein. The protein measurement of the blank would then be subtracted from the measurement of the whole reaction.

Another approach would be to use a different protease assay. There is a version of the casein assay that uses fluorescein-labeled casein. The fluorescein labeling is quite dense, leading to self-quenching of fluorescence. Proteolysis releases fluorescein-labeled peptides, relieves the quenching, and results in a fluorescence increase. This can be measured using a fluorescence plate reader or spectrofluorometer. Since this assay measures fluorescence intensity rather than protein concentration, there is no interference from the peptidic protease inhibitors. Another advantage is that you don't have to separate the undigested casein from the released peptides.

If you are using a specific protease, you can also use a synthetic chromogenic or fluorogenic peptide substrate appropriate to that protease. The peptidic inhibitors would not interfere with this method either. The chromogenic substrates would allow you to use a spectrophotometer or absorbance plate reader.

DEar Stephanie

ifi i well understand what do you would like to check, as also suggested from Adam, you can use a kit as the following:

https://www.fishersci.com/shop/products/pierce-protease-assay-kits-1/PI23267

which contain fluorescence casein and compare the rate of casein digestion (that could be monitored with colorimetric or fluorometric approach) in presence of different amount of inhibitors.

ciao

Manuele

Many thanks to both of you for the information you have provided - I appreciate your input very much.