Protein Expression and Purification Series - Bio-Rad

CHAPTER 9
DHFR ENZYMATIC
ASSAY
Protein Expression and Purification Series
3. Blanking the instrument at 340 nm using 1x PBS
a. If you have a spectrophotometer that can be programmed to take multiple readings at a specified
time interval (kinetics mode), program the instrument to take readings at 340 nm every 15 seconds for
150 seconds.
b. If you do not have a kinetics mode on your spectrophotometer, set the instrument to read at
340 nm and have a stopwatch handy to measure time intervals.
c. Fill a UV compatible cuvette with 985 µl of 1x PBS.
d. Insert the cuvette into your spectrophotometer and press the key that will blank the instrument at
340 nm so that 1x PBS gives a reading of 0.000 at 340 nm.
e. Take your cuvette out of the spectrophotometer and save it and the 985 µl of 1x PBS for step 4.
4. Running the no substrate control reaction
You will now add your enzyme sample of GST-DHFR-His with DHFR’s cofactor, NADPH, to the
1x PBS. Since there is no substrate (DHF) being added to this control reaction, the absorbance at 340 nm
contributed by the NADPH should not change since it is not being converted to NADP+.
a. Add 6 µl of 10 mM NADPH to the cuvette already containing 985 µl of 1x PBS.
b. Add 15 µl of your chosen GST-DHFR-His sample to the cuvette.
c. Cover the cuvette with parafilm and invert 10 times to fully mix the GST-DHFR-His and NADPH into
the 1x PBS.
d. Place the cuvette into your spectrophotometer and measure the absorbance at 340 nm every 15
seconds for 150 seconds (either manually while timing with a stopwatch or using the kinetics mode if
your instrument has one).
Note: If you do not have a printer on your spectrophotometer, record the absorbance values you
measure every 15 seconds in your notebook.
e. If you have a printer on your spectrophotometer, after the 150 seconds of readings have been
completed, print out your data and label it “No Substrate Control Reaction.”
f. Remove your cuvette from the spectrophotometer and save it with all of its contents to be used for
running the enzymatic reaction.
5. Running the enzymatic reaction with GST-DHFR-His, NADPH (cofactor) and
DHF (substrate)
Note: The enzyme reaction should be prepared while standing at the spectrophotometer. The reaction
occurs extremely quickly (within seconds) and it is necessary to mix very quickly, place the cuvette in
the spectrophotometer, and begin taking readings as quickly as possible after you have added the DHF
substrate.
a. Make sure that your spectrophotometer is still programmed to take readings at 340 nm (in kinetics
mode if available).
204 Chapter 9: DHFR Enzymatic Activity Assay Student Protocol