Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
DHFR Enzymatic Activity Assay
Determining which fraction to test for enzyme activity.
Review your SDS-PAGE gel from your purification to determine which fraction(s)
to test. Note that centrifugation purification will have one desalted fraction while
instrumentation-based purification will have three fractions.
Set up spectrophotometer for kinetics measurements at
340 nm.
Blanking the instrument. Add 985 µl 1x PBS to cuvette;
place in instrument, read as blank. Save cuvette with PBS.
Running the no substrate control reaction.
Add 6 µl of 10 mM NADPH to cuvette containing 985 µl 1x PBS.
Add 15 µl of purified, GST-DHFR-His eluate to cuvette. Cover
cuvette with parafilm and invert 10 times to mix
Place cuvette in spectrophotometer and begin kinetics run.
As run is proceeding, record absorbance value every
15 seconds for 150 seconds. Remove and save cuvette from
the spectrophotometer.
Running the enzymatic reaction with the 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 and it is necessary to place the cuvette in the
spectrophotometer and start the readings as quickly as
possible once the DHF has been added.
Add 5 µl of 10 mM DHF to the cuvette already containing
1x PBS, your GST-DHFR-His sample and NADPH. Quickly
cover the cuvette with parafilm and invert five times.
Place the cuvette in the spectrophotometer and begin kinetics
run. As run is proceeding, record absorbance value every
15 seconds for 150 seconds. Remove cuvette from the
spectrophotometer.
Calculate the activity following the instructions in the manual.
Chapter 9: DHFR Enzymatic Activity Assay Student Protocol
209
+ 985 µl
1x PBS
+ 15 µl
GST-DHFR-His
eluate
1x PBS
1x PBS
NADPH
GST-DHFR-His eluate
1x PBS
NADPH
GST-DHFR-His eluate
+ 6 µl 10
mM
NADPH
+ 5 µl
10 mM DHF
CHAPTER 9
DHFR ENZYMATIC
ASSAY