Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
4.
Look at the front panel of the BioLogic LP system and record the final conductivity and absorbance
values after pure Buffer B has been run through the system.
Absorbance ________________ AU
Conductivity _______________mS
Buffer B has been running through the system, and it contains high salt and imidazole levels which
contribute to a higher conductivity reading. The conductivity readings after equilibrating with Buffer
B should be approximately 35–55 mS. Imidazole also absorbs at 280 nm so there should be some
absorbance due to the 250 mM imidazole in Buffer B. The absorbance should be approximately
0.00–0.15 AU.
Now the column will be primed with Equilibration buffer (98% Buffer A, 2% Buffer B = 20 mM
Phosphate buffer, 300 mM NaCl, 5 mM Imidazole) in preparation for binding of the GST-DHFR-His.
5. Press the BUFFER softkey and then press the MIX softkey.
Note: MIX is a mixture of Buffer A and Buffer B.
6. Using the number keypad, enter 2% B then press the OK softkey.
7. Press the START softkey to begin flow. Allow the system to run for seven minutes (14 ml of
Equilibration Buffer). You will hear a clicking sound which is the proportioning valve/mixer pulling from
both beakers of buffer.
8. After seven minutes, press the STOP softkey to stop the flow.
9.
Note the final conductivity and absorbance values after 2% Buffer B has been run through the system.
Absorbance ________________ AU Conductivity _______________mS
Only 2% Buffer B has been running through the system and it contains high salt but low imidazole levels.
The high salt contributes to a higher conductivity reading (expected range of 35-55 mS). However, in
comparison to 100% Buffer B, only 2% Buffer B should have little to no absorbance at 280 nm (in the
range of 0.000–0.05 AU).
Select your Fraction Collector
BioFrac fraction collector only—fraction collector setup
Note: If you are using a Model 2110 fraction collector, proceed to the Model 2110 Fraction Collector setup
on the next page.
1.
Turn the BioFrac fraction collector’s power switch On. The switch is in the back right hand side of the
instrument.
On the BioFrac fraction collector front panel, using the
2. Cursor Control Arrow keys, move the flashing
cursor until it is at Mode: options. Press the ENTER key to see the options. Use the Cursor Control
Arrow keys to select LP/Econo and then press the ENTER key again.
Chapter 7: Purification Protocol for BioLogic LP System
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CHAPTER 7
BIOLOGIC LP SYSTEM
PROTOCOL