Protein Expression and Purification Series - Bio-Rad

CHAPTER 3A
ADVANCE PREP
CENTRIFUGATION PROCES
Protein Expression and Purification Series
Advanced Preparation for the DHFR Enzymatic Assay
In the DHFR enzymatic assay, the enzymatic activity of the purified, desalted GST-DHFR-His is measured
spectrophotometerically by detecting the decrease in absorbance at 340 nm over time of the NADPH
cofactor as it is converted to NADP + .
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 12 ❒
100–1,000 µl adjustable-volume micropipets and tips 12 ❒
UV spectrophotometer capable of three decimal place accuracy 1–2 ❒
trUView disposable cuvettes (or UV compatible cuvettes) 12 ❒
Parafilm 12 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 begins, 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 begins, 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.
Aliquot 10 µl DHF substrate into a 2 ml microcentrifuge tube for each workstation.
Note: Make sure and use 10x PBS to reconstitute the 1 mg of DHF. The high salt in 10x PBS helps
dissolve the DHF.
4. Turn on the spectrophotometer at least 30 minutes before the lab period 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 that came with the instrument to
determine if kinetics mode can be programmed and how to set the wavelength to 340 nm. If there is
no kinetics mode, readings at 340 nm can be taken manually every 15 seconds for 150 seconds and
recorded.
Note: UV compatible cuvettes (trUView or quartz) that can read 1 ml samples must be used for this
activity.
70 Chapter 3A: Advance Preparation
for Centrifugation Protocols