Troubleshooting a Protease Activity Assay - Why r my halos coming out blue instead of clear? HELP A PORR STUDENT
Hi everyone, I’m a Master's student studying leaf senescence and I’m sufferying trying to estimate protease activity using an agar diffusion assay, but I’ve hit a wall.
So, the method was simple:
- Medium: 1.5% Agar enriched with 0.1% BSA.
- Procedure: I pipette a protein-rich extract (lyophilized leaves in phosphate buffer, normalized to 0.1 mg of protein) into small pits in the agar.
- Incubation: 24 hours at room temperature.
- Staining: 2 hours with Coomassie Brilliant Blue (CBB), followed by a destaining solution.
The Problem: In my head (and my supervisor’s), the proteases should digest the BSA, leaving a colorless/clear halo around the wells. Instead, I’m getting the exact opposite: darker blue halos around the pits.
What I’ve tried so far:
- Adjusting the exposure time for both the Coomassie stain and the destainer. (not better)
- Moving to a colorimetric method using Azocasein, but the substrate doesn't seem to dissolve completely and is very difficult to work with.
I feel like I'm the only person I know doing this specific method for coffee leaves. Has anyone dealt with these "reverse halos" before? Could it be protein-protein interference or the high concentration of my extract proteins staining more than the BSA?
Any advice on how to fix this or a recommendation for a more reliable protease assay for plant tissue would be a lifesaver!