Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series 1. 2. 3. Press the Settings button on the top menu bar. Select the parameters that you want to be able to track on the Manual screen chromatogram by scrolling down the Trace Device options, selecting an option, and then clicking on the Visible box (Figure 8.10). If there is an “X” in the visible box then that trace can be displayed. Select UV, Conductivity, GP-Pressure and % Buffer B to be visible, and then click OK. 1.8 Status bar At the bottom of the Manual screen is a status bar that is continually updated with system parameters (Figure 8.11A). 1. 2. 3. 4. 5. Allow the pumps to run for three more minutes. Press the Zero Baseline button on the UV control (Figure 8.8). Figure 8.10. Controlling the chromatogram settings. The traces that will be possible to scroll through on the Manual screen chromatogram are selected to be visible under the Settings option. The maximum value for the axis can also be set here or by using the scroll bars on the actual chromatogram (Figure 8.9). Press the Clear Traces button (Figure 8.9) to clear the Manual screen chromatogram. At this point, you have primed the entire system and rinsed it out with water. Record the UV and conductivity values from the status bar after the water wash. UV _______________________________ Conductivity ________________________ mS/cm Ideally, the UV should be close to zero since high-quality water has minimal to no absorbance at 280 nm. The conductivity should also be close to zero. The conductivity depends on how many ionic species are present. HPLC grade water such as nanopure water, distilled or deionized water should have a conductivity of less than 3 mS/cm. If the UV value and conductivity values are higher than 0.1, continue running high-quality water through the system for another five minutes 172 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series A B Figure 8.11. Manual screen chromatogram and status bar after five minutes of water washing at 2.00 ml/min. A) This is a magnified view of the status bar. The flow rate is displayed, followed by the % B and the pressure. The UV and conductivity readings are close to zero which means that the system is filled with water and has been cleaned. B) The Manual screen chromatogram shows the UV and conductivity readings for the five minutes of water cleaning time. Note that the Y-axes have been greatly reduced with the AU (UV readings), ranging from -0.005 to 0.0025, and the conductivity readings ranging from 0.000-0.150 mS/cm. Within the accuracy of the detectors, there is little to no change in the conductivity or UV readings for the five minutes, and the values are close to zero. Chapter 8: Purification Protocol for BioLogic DuoFlow System 173 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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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 5 100 ml CULTURE PROTOCOL P
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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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