Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Quantitation of Protein in Desalted Fractions
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 fractions (150 µl each) 3
20–200 µl adjustable-volume micropipet and tips 1
trUView disposable cuvettes (or UV compatible cuvettes) 3
Marking pen 1
Common Workstation Quantity
UV spectrophotometer 1–2
Protocol: Quantitation of Protein in Desalted Fractions
1.
2.
Make sure that your cuvettes are completely clean and dry before use.
Set your spectrophotometer to read at 280 nm.
Note: If using a Bio-Rad SmartSpec Plus spectrophotometer, see Appendix D for setup instructions.
3. Blank your spectrophotometer with 100 µl distilled water.
4. Pipet 100 µl of your desalted fractions A, B, and C each into its own clean cuvette.
5.
6.
Measure the absorbance at 280 nm of all of your desalted fractions.
A 280 Desalted fraction A __________________________
A 280 Desalted fraction B __________________________
A 280 Desalted fraction C __________________________
Pipet each desalted fraction back into the tube containing that fraction after measuring its absorbance.
7. Make sure that you have at least 15 µl of desalted GST-DHFR-His to run your enzyme assay.
8.
Calculate the concentration of GST-DHFR-His in your desalted fractions 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
Chapter 8: Purification Protocol for BioLogic DuoFlow System
191
CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL