Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Figure 8.2. Manual control screen with UV detector. This screen allows manual control of the gradient pump (flow rate, % Buffer A/B, high and low pressure limit for the pump), control of the Fraction collector, the UV detector, and the workstation valves. On the bottom of the screen is a chromatogram that can display all variables that are being monitored such as UV, conductivity, pressure, and % Buffer B. The real time display of the gradient pump flow rate, buffer composition, pressure reading, UV reading and conductivity reading are displayed on the bottom line of the manual control page. 4. 5. 6. 7. Turn the priming port counterclockwise one full turn to open the seal. Gently withdraw the syringe plunger to draw 10 ml of water into the pump head and tubing. Look at the inlet tubing that goes from the beaker of water to Pump A. If there are any air bubbles in the tubing, tighten the priming port by turning it clockwise, empty the syringe, and repeat steps 3–5. Tighten the priming port by turning it clockwise and then remove the syringe. Repeat this priming procedure for pump B. Figure 8.3. Priming the workstation pumps. A complete pump head on the DuoFlow system is shown here. Priming pump: A syringe is used to draw fluid into the inlet tubing for Pump A and Pump B. The pumps cannot pull fluid into the lines if there is air in the lines. Priming also serves to remove any air bubbles from the lines that could damage columns further downstream. 168 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series 1.2 Move the AVR7-3 inject valve to the purge position Prior to purging the pumps at 10 ml/min it is essential to place the AVR7-3 valve in the purge position. This directs the flow to waste and not to the column and detectors. 1. 2. To change the position of the AVR7-3 inject valve select “P” from the manual screen valve control panel for the AVR7-3 valve (Figure 8.4B). Look on the AVR7-3 valve itself. The “P” should be showing on the valve between ports 1 and 2 (Figure 8.4B). Figure 8.4. Workstation valve manual controls. A) The default setting upon startup of the BioLogic DuoFlow system for the AVR7-3 valve is L (Load). This valve gets its name from the fact that there are seven ports in total and of those, three are inlet ports. One of the three settings that the AVR7-3 valve can be set for is L (Load) in which a sample can be loaded into the sample loop with any excess solution from the sample loop being pushed to waste. At the load setting, the pump pushes fluid into the AVR7-3 valve and out a second port without pushing any of the sample in the sample loop further along the fluid pathway onto the column. The I (Inject) position is where the valve moves so that now the fluid pushed through the pumps pushes the sample out of the sample loop and injects it onto the column. B) The P (Purge) setting allows fluid from the pumps to be pushed into the AVR7-3 valve and then out a waste port. In purge mode, the fluid from the pumps does not push the sample in the sample loop onto the column and in fact, does not even continue to flow buffers from the pumps any further along the fluid pathway. The purge setting is used to remove or exchange buffers in the tubing bypassing the column at a high flow rate that the column potentially cannot A B withstand. The AVR7-3 is in purge mode as shown by the “P” on the valve. 1.3 Purge the workstation pumps Purging of the workstation pumps flushes out any storage buffer or remaining solutions that are in the tubing between the buffer inlets and the AVR7-3 valve. This serves to clean out the tubing and also replace whatever buffer was in the tubing with water. 1. 2. 3. 4. Press the Purge button A on the front of the workstation (Figure 8.5) and allow it to run for two minutes. The green light next to pump A should be flashing green. Press the Purge button A on the front of the workstation a second time to stop pump A. Press the Purge button B to start this pump and allow it to run for two minutes. The green light next to pump B should be flashing green. Press the Purge button B again to stop the pump. Chapter 8: Purification Protocol for BioLogic DuoFlow System 169 CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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CHAPTER 1 BACKGROUND Protein Expres
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CHAPTER 2 FOCUS QUESTIONS Protein E
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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CHAPTER 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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APPENDIX C RCF TO RPM CONVERSTION P
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX D SETTING UP THE SMARTSPEC
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APPENDIX E MINI-PROTEAN GEL BOX SET
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX F BIOLOGIC LP SYSTEM SETUP
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX G BIOMANUFACTURING Protein
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APPENDIX H FOCUS QUESTIONS INSTRUCT
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APPENDIX I GLOSSARY Protein Express
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APPENDIX I GLOSSARY Protein Express
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APPENDIX J REFERENCES, LEGAL NOTES
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Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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