Protein Expression and Purification Series - Bio-Rad

CHAPTER 4
11 ml CULTURE
PROTOCOL
Protein Expression and Purification Series
Quantitation of Protein in Desalted Fraction
Student Workstations
Each student team requires the following items to quantitate the amount of purified, desalted eluate
(GST-DHFR-His) they have via spectroscopy:
Material Needed for Each Workstation Quantity
Desalted eluate sample 150 µl
20–200 µl adjustable-volume micropipet and tips 1
trUView disposable cuvette (or similar UV compatible cuvettes) 1
Marking pen 1
Common Workstation Quantity
UV spectrophotometer 1–2
Protocol: Quantitation of Protein in Desalted Fraction
1. Make sure that your cuvettes are completely clean and dry before usage.
2. Set your spectrophotometer to read at 280 nm
Note: If using a Bio-Rad SmartSpec Plus spectrophotometer, please see Appendix D for setup
instructions.
3. Blank your spectrophotometer with 100 µl distilled water.
4. Pipet 100 µl of your desalted eluate sample into a clean cuvette.
5. Measure the absorbance at 280 nm of your desalted eluate sample.
A 280 Desalted eluate sample: __________________________
6. Pipet the sample from the cuvette back into your tube of desalted eluate sample. Make sure that you
have at least 15 µl of desalted eluate sample to run your enzyme assay.
7. Calculate the concentration of GST-DHFR-His in your desalted eluate fraction as follows:
A. The extinction coefficient (ε) of the entire GST-DHFR-His construct is theoretically calculated to be
75,540 M -1 cm -1 .
Knowing that Absorbance = ε x C x L,
where ε is 75,540 M -1 cm -1
L is the pathlength of the cuvette in cm (usually 1)
and the absorbance at 280 nm is being measured
The concentration of GST-DHFR-His (M) = Absorbance/75,540
Concentration of GST-DHFR-His = ______________________ M
104 Chapter 4: 11 ml Culture Protocol
for Centrifugation Purification