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- Choose a working protein concentration of around 5–10 μg/
mL or an optimized concentration from an earlier activity assay. - Dilute the protein into 2× Assay Buffer to a fi nal concentration
of 2× the chosen working concentration (~10–20 μg/mL). - Place a 96-well black-bottomed plate on ice.
- Add 100 μL ddH 2 O to 11 wells on single row (wells 1–11) of
the plate. - In well 12 add 200 μL of substrate, diluted to 200 μM in
ddH 2 O. - Using a pipette, remove 100 μL of the solution in well 12 and
place into the adjacent well (well 11). Mix by gently pipetting. - Repeat this 2-fold (1/2) dilution (transferring 100 μL from
well 11 to well 10 and so on) for all remaining wells. - Discard 100 μL of the diluted substrate in well 1, leaving
100 μL substrate in each well. - Keeping the plate on ice, add 100 μL of the 2× protein–buffer
mixture to each well. - Remove the plate from the ice, place in the fl uorescence plate
reader and equilibrate to ambient temperature (20–25 °C) for
5 min. - Read the fl uorescence using an excitation wavelength of
355 nm and emission wavelength of 460 nm, using time scales
and intervals described above. - Plot the FU versus T for each substrate concentration.
- Calculate the reaction rate (FU/ T ) for each substrate concen-
tration, as above. - Plot substrate concentration versus reaction rate. This should
give a hyperbolic plot, where the y value approaches V max. If
the plot is not hyperbolic, the substrate range used in the assay
may need to be altered. - Plot 1/[substrate] versus 1/reaction rate (Lineweaver–Burk
plot). The slope of the fi tted line is equal to 1/ Km. Alternatively,
a program such as GraFit or Prism can be used to automati-
cally calculate Km. - Prepare protein by concentrating or diluting to between 200
and 400 μg/mL in 1× Assay buffer. This is a 40× working
solution, giving a fi nal assay concentration of 5–10 μg/mL. - Dilute the inhibitor to 100 μM in ddH 2 O (stock inhibitor)
and place a 96-well black-bottomed plate on ice. - Dilute a fl uorogenic substrate to 100 μM in ddH 2 O, preparing
enough for 20 μL per well.
3.5.4 Calculation of Km
3.5.5 Calculation of IC 50 s
for Inhibitors
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