Michaelis-Menten Assay

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

Make one for each enzyme

• HEPES 7.4 pH (250mM)
• 1% Tween
• Purified Enzyme (50nM)
  • Dilute enzyme in 1x HEPES/Tween buffer

Note: Make one without enzyme for blank

Prepare Substrate

• Prepare 10x substrate (10uM) in one well of a 12-well strip tube
• Do half concentration serial dilutions (equal parts diH2O and substrate) until 11th well.
• Leave the 12th well with no substrate

Final Concentrations of Substrate (Left to Right in uM):

10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.01953125, 0.009765625

1.1x Master Mix

• Put equal parts 10x Master Mix and 10x L-Glu eppendorf tube (Referred to as L-Glu Master Mix)
• Put equal parts 10x Master Mix and diH2O in an eppendorf tube (Referred to as diH2O Master Mix)
• Fill each tube with diH2O (70uL/rxn)

Prepare a 96-well plate for transpeptidation

• Pipette 90uL of 1.1x L-Glu Master Mix for one each enzyme across a row for the transpeptidation reaction
• Repeat for each enzyme
• Make one row of blank (1.1x L-Glu Master Mix without enzyme)

Prepare a 96-well plate for hydrolysis

• Pipette 90uL of 1.1 diH2O Master Mix for each different enzyme in each new row
• Repeat for each enzyme
• Make one row of blank (1.1x diH2O Master Mix without enzyme)

Prepare Spectramax plate reader with desired settings

• Pipette 10uL of Substrate into each well. (Column 1 from well 1 of strip tube, Column 2 from well 2 of strip tube, etc.)
• 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 - Variable Concentration
• HEPES (7.4pH) - 25mM
• 0.1% Tween

Final Reaction Volume 100uL

Substrate is 5-FAM-(D-gamma-Glu)5-K(QXL520-NH2 from AnaSpec

Product produced from reaction is 5-FAM-(D-gamma-Glu)5-NH2