I am determining protease activity in outer membrane vesicles (OMVs) isolated from clinical gram-negative bacteria. I prepared a 0.5% azocasein solution in 0.5M Tris buffer at pH 7, gently mixing it at 40°C for 5 minutes. I then added 100 µg/ml of OMVs to 200 µl of the azocasein solution and incubated the mixture for 2 hours at 37°C. To stop the reaction, I added 10% trichloroacetic acid (TCA) and incubated it for 30 minutes at room temperature. After centrifugation, I collected the supernatant in a new microcentrifuge tube, added 1N NaOH, and measured the absorbance at 450 nm. My negative control contained only the azocasein solution and PBS without enzymes, while the positive control included 20 µl of Proteinase K with the substrate.