Protein Expression and Purification Series - Bio-Rad

CHAPTER 8
BIOLOGIC DUOFLOW
PROTOCOL
Protein Expression and Purification Series
3.
4.
Choose Isocratic Flow from the left-side menu. Change the composition of the 2x PBS (Buffer A) to
98%. The % Composition for Buffer B will automatically change to 2%. Change the volume to 5.00 ml
and the flow rate to 2.00 ml/min, and then press OK (Figure 8.25).
Figure 8.25. Programming Step 3: Equilibrating the column with 2x PBS + 5 mM imidazole for 5 CV at 2 ml/min.
Continue programming steps 4–9 as defined in Table 8.2 below. Your final protocol should look like
Figure 8.26.
Table 8.2. Programming the Protocol
Step Number Start (ml) Purpose Programming
1 0.00 Programming fraction
collector
2 0.00 Turn the lamp on
(if not already on)
Collect 4.00 ml fractions during the
sample injection and wash steps and then
1.00 ml fraction during the elution step
Lamp
Choose on
3 0.00 Equilibrate column Isocratic flow with 98% 2x PBS and 2%
2x PBS, 250 mM Imidazole for 5 ml at
2.00 ml/min
4 5.00 Zero baseline Zero baseline to set UV baseline to 0.0
5 5.00 Inject sample onto the
column
Load Inject sample, static loop: Inject 6.0
ml* sample at 1.00 ml/min. The injection
occurs using 98% 2x PBS and 2% 2x
PBS, 250 mM Imidazole
6 11.00 Wash the column Isocratic flow with 96% 2x PBS and 4%
2x PBS + 250 mM Imidazole for 11 ml at
2.00 ml/min
7 22.00 Elute GST-DHFR-His Isocratic flow with 0% 2x PBS and 100%
2x PBS, 250 mM Imidazole, for 8 ml at
2.00 ml/min
8 30.00 Final column wash Isocratic flow with 100% 2x PBS and 0%
2x PBS, 250 mM Imidazole for 15 ml at
2.00 ml/min
9 45.00 Turn off lamp Lamp Choose off
45.00 End of protocol
*A slightly larger volume (6 ml) than the sample loop (5 ml) is used to ensure all the sample is fully pushed onto the column.
182 Chapter 8: Purification Protocol for BioLogic DuoFlow System