Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Final Check Before Starting Method
1.
2.
3.
4.
Check for leaks or air in the lines.
Check to make sure that your buffer inlet lines are at the bottom of the beakers containing Buffer A
and Buffer B.
Make sure that you have at least 50 ml of Buffer A and 20 ml of Buffer B.
Place collection tubes in the fraction collector rack in the first two columns (30 tubes total) for the
BioFrac fraction collector or the first 30 spaces for the Model 2110 fraction collector.
5. If you are using LP DataView software, press STOP on the software menu. Press CLEAR to clear
all previous information. A menu box will appear. Choose “NO.” Press RECORD to record your
purification run.
Load Sample Loop with GST-DHFR-His Soluble Fraction
1.
2.
3.
4.
5.
Make sure the MV-6 injector valve knob counter is turned
counterclockwise (left) as far as it will go (Figure 7.10).
Draw 5 ml of the soluble fraction of GST-DHFR-His into the 10 ml
syringe. Avoid pulling in any air bubbles. If you have more than 5 ml of
soluble fraction, you can draw the entire fraction into the syringe.
Make sure to have the waste port tubing of the MV-6 injector valve
over a waste beaker.
Insert the syringe in the top port of the MV-6 injector valve and fill the
sample loop. The excess water that was in the sample loop from the
cleaning of the loop will be pushed out of the waste port.
Note: Avoid pushing air bubbles into the sample loop since these will
flow into the column and through the detectors.
Leave the syringe in the port when you have injected the sample; this prevents the sample from
siphoning out of the loop.
Start Method
1. Press the Run mode key.
2. Immediately turn the injector valve knob clockwise
(to the right) as far as it will go (Figure 7.11).
Figure 7.11. MV-6 injection port in the INJECT position. The
inject position (turned fully clockwise) opens up the fluid pathway
so that the buffer coming from the pump will push the sample out
of the sample loop and onto the column.
Chapter 7: Purification Protocol for BioLogic LP System
149
Figure 7.10. MV-6 in Sample
Load position, turned as far left
as possible.
CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL