Protein Expression and Purification Series - Bio-Rad

APPENDIX B
RESULTS ANALYSIS
Protein Expression and Purification Series
Step 5: Check that desalting of GST-DHFR-His was successful.
Lane 9 is the desalted GST-DHFR-His eluate fraction.
Results: There should be a band of approximately 43 kD that accounts for the majority of the protein in lane
9 that is of comparable size to the main band in lane 8 and about the same darkness. The absorbance
value of this fraction at 280 nm should give a significant value above the blank absorbance.
Step 6: Check that the purified GST-DHFR-His has enzymatic activity.
Results: The “No Substrate Control Reaction” should show a fairly flat line with an absorbance around
0.2–0.4 (Figure 2, below). The enzyme reaction should show a decrease in absorbance at 340 nm over
the 150 seconds of reading time (Figure 3, below). The calculated reaction rate will vary depending on the
amount of GST-DHFR-His purified.
Absorbance at 340nm
Figure 2. No substrate control enzymatic activity reaction.
Absorbance at 340nm
Figure 3. GST-DHFR-His enzyme activity reaction.
No Substrate Control Reaction
Time in seconds
GST-DHFR-His Enzyme Reaction
Time in seconds
214 Appendix B: Purification Results Analysis