Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Advance Preparation for the DHFR Enzymatic Assay
In the DHFR enzymatic assay, the enzymatic activity of the purified, desalted GST-DHFR-His is measured
spectrophotometrically by detecting the decrease in absorbance at 340 nm over time of the NADPH
cofactor as it is converted to NADP + .
Note: The following quantities are based on assaying the desalted GST-DHFR-His fraction with the highest
concentration and darkest band on the SDS-PAGE gel that also appears to be the most pure fraction
from the SDS-PAGE analysis. There are enough reagents (1x PBS, NADPH and DHF) for each of the four
student workstations to perform three enzymatic reactions for each station, one for each of three desalted
fractions. The quantities listed below for the student workstations would need to be adjusted accordingly.
DHFR Enzymatic Reaction Assay Checklist
Components from Protein Expression
and Purification Series Where Provided (✔)
10x PBS Growth and Expression Module ❒
NADPH cofactor DHFR Enzymatic Assay Module ❒
DHF substrate DHFR Enzymatic Assay Module ❒
Required Accessories (Not Provided) Quantity (✔)
2–20 µl adjustable-volume micropipets and tips 4 ❒
100–1,000 µl adjustable-volume micropipets and tips 4 ❒
UV Spectrophotometer capable of three decimal place accuracy 1–2 ❒
trUView disposable cuvettes (or UV compatible cuvettes) 4–12 ❒
Parafilm 4 squares ❒
Tasks to Perform Prior to the Lab
1. Prepare 15 ml of 1x PBS by combining 13.5 ml of distilled water with 1.5 ml of 10x PBS and mix
thoroughly.
2. Immediately before the laboratory exercise is to be run, reconstitute the 1 mg of NADPH by adding
120 µl of 1x PBS to the vial of NADPH and invert or vortex to mix and ensure complete dissolution.
The concentration of the dissolved NADPH is 10 mM. Store the NADPH on ice until ready to use.
Once reconstituted, the NADPH solution is only stable for 3–4 hours. The NADPH solution cannot be
frozen to retain or extend the activity and shelf life.
Aliquot 8 µl of NADPH cofactor into a 2 ml microcentrifuge tube for each workstation.
3. Immediately before the laboratory exercise is to be run, reconstitute the 1 mg of DHF by adding
226 µl of 10x PBS to the vial of DHF and invert or vortex to mix and ensure complete dissolution.
The concentration of the dissolved DHF is 10 mM. Store the DHF on ice until ready to use. Once
reconstituted, the DHF solution is only stable for 3–4 hours. The DHF solution cannot be frozen to
retain or extend the activity and shelf-life.
Note: Make sure to use 10X PBS to reconstitute the 1 mg of DHF. The high salt in 10x PBS helps
dissolve the DHF.
Aliquot 10 µl DHF substrate into a 2 ml microcentrifuge tube for each workstation.
4. Turn on the spectrophotometer at least 30 minutes before the lab to allow the lamp to warm up.
If using the Bio-Rad SmartSpec Plus spectrophotometer, step-by-step instructions are available
in Appendix D for setting up the instrument in kinetics mode to read at 340 nm. If using another
manufacturer’s spectrophotometer, consult the instruction manual for that instrument to determine if
Chapter 3B: Advance Preparation for
Chromatography Instrumentation Protocols
89
CHAPTER 3B
ADVANCE PREP
INSTRUMENTATION PROCES