Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Chapter 8: Purification Protocol for BioLogic DuoFlow System Affinity Purification of GST-DHFR-His Using a BioLogic DuoFlow System Student Workstations Each student team requires the following items to purify their GST-DHFR-His samples using affinity chromatography: Material Needed for Each Workstation Quantity Soluble lysate fraction 5–7.5 ml 10 ml syringe 1 Distilled water 500 ml Buffer A 70 ml Buffer B 40 ml Collection tubes for fraction collector 32 Marking pen 1 Common Workstation Quantity BioLogic DuoFlow chromatography instrument with 5 ml sample loop and column installed: Either a BioScale Mini Profinity Ni-IMAC Cartridge or 1–4 A glass Econo-Column column handpacked with 1 ml of Profinity IMAC Ni-charged resin BioLogic DuoFlow injection needle 1–4 Preparing the DuoFlow system for Use 1. Turn on the power switches for all hardware (e.g. Maximizer, if you have one), Workstation, and BioFrac fraction collector. Turn on the power for the Controller (computer) once all the other hardware has been turned on. 2. 3. Double click on the BioLogic Configuration icon. Make sure that the configuration is set up for the system that you have installed (Figure 8.1), then click “OK” to close out the Configuration Utility. 4. Double click on the BioLogic DuoFlow application icon to launch the software. 166 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series Should be checked if a Maximizer is being used; unchecked if no Maximizer is being used. Check which pump heads are installed and choose the correct one. Figure 8.1. BioLogic System Configuration Utility. This utility is used to communicate the configuration of the hardware installed with the BioLogic DuoFlow Controller software. If a Maximizer is installed, the box should be checked. Next, the type of fraction collector should be checked. Finally, the pump head size should be highlighted—F10 for the pump heads that can produce 10 ml/ min maximum flow and F40 for the pump heads that can produce 40 ml/min maximum flow. Section 1. DuoFlow System Preparation When the DuoFlow system is turned on, the Manual screen is displayed (Figure 8.2). This screen displays instrument control panels that provide direct control of the pumps, valves, fraction collector, and UV detector. Only those valves and accessories connected to the system will be displayed. 1.1 Prime the workstation pumps Priming the workstation pumps involves filling the buffer inlet tubing with water. If there is air or air bubbles in the inlet lines, the pumps cannot pull fluid efficiently. Priming the pumps also serves as a first rinse of the inlet lines to remove any storage buffers that might be present. 1. 2. 3. Make sure that the waste lines of the AVR7-3 and the SV3-2 valve for the BioFrac fraction collector are flowing into a waste container. Immerse the workstation pump A and B inlet lines in a bottle containing 500 ml of HPLC grade (filtered, degassed) or other high-quality water. Connect a clean 10 ml syringe to the priming port of pump A (Figure 8.3). Chapter 8: Purification Protocol for BioLogic DuoFlow System 167 Click OK when changes are complete. If you are using a BioFrac fracton collector, make sure that the circle is darkened here. 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 B RESULTS ANALYSIS Protein
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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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