Enzyme Assay

Run one transpeptidase reaction and one hydrolysis reaction per enzyme (always run control for each set)

Prepare 10x Master Mix with these concentrations. (10uL/rxn)

• HEPES 7.4 pH (250mM)
• 1% Tween
• Substrate (500nM)

Dilute Enzymes

• Measure enzyme concentrations by protein gel band intensity or with spectrophotometer (A280)
• Dilute enzymes to 50nM (~0.0025mg/mL) in strip tubes
  • Make sure you have at least 30uL of your final diluted enzyme

Prepare a 96-well plate

• Pipette diH2O into each well (Two per enzyme)
  • Transpeptidase: 70uL
  • Hydrolysis: 80uL
• Pipette 10uL of 10x Master Mix into each well.
• Pipette 10uL of 50mM L-Glu into each transpeptidation reaction well.

Prepare Spectramax plate reader with desired settings and desired emission and excitation wavelengths

• Pipette 10uL of your enzyme into each well
• Immediately place into Spectramax plate reader and begin reading

Final Concentrations

• Enzyme (CapD) 5nM (0.00025mg/mL)
• Amino Acid (L-Glutamate)0mM or 5mM
• Substrate 50nM
• HEPES (7.4pH) 25mM
• 0.1% Tween
• Final Reaction Volume 100uL