Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Figure 8.22. Fraction Collection Scheme programming. This window allows programming of the BioFrac fraction collector. The options are: Collect All, where all fractions are collected and each has the same volume; Threshold, where fractions are collected if the absorbance is greater than a set value; Collection Windows, where fractions of differing volume are collected in different time frames (windows); and Threshhold & Collection Windows, where fractions collected during certain time frames have an absorbance greater than a set value. Collection Windows will be used for the GST-DHFR-His protocol. Larger volume fractions will be collected for proteins that do not bind to the column (flowthrough fraction) and are washed off the column (wash fractions) while smaller fractions will be collected when the GST-DHFR-His elutes. This allows for more concentrated GST-DHFR-His samples. 3. 4. 5. Program the first window by typing 6.00 into the Start (ml) box, 12.00 into the End (ml) box and 4.00 into the Frac. Size (ml) box. Press the Save Window button. Table 8.1 Programming BioFrac fraction collector collection windows. Step Number Start (ml) End (ml) Frac. Size (ml) Product being collected 1. 6.00 12.00 4.00 Flowthrough 2. 12.00 23.00 4.00 Wash 3. 23.00 37.00 1.00 Eluted GST-DHFR-His Press the Add Window button. Enter 12.00 for the Start (ml), 23.00 for the End (ml) and 4.00 for Frac. Size (ml). Press the Save Window button. Press the Add Window button. Enter 23.00 for the Start (ml), 37.00 for End (ml) and 1.00 for Frac. Size (ml). Press the Save Window button, then Close. Programming the pump and UV lamp steps of the protocol 1. Program the remaining steps using the Add Step icons on the left side of the screen. Note that Step 1 has already been added to your protocol since you already programmed the fraction collector. Table 8.2 on page 182 includes all of the remaining steps that need to be programmed. Examples of how to program Steps 2 and 3 are outlined here. Note: The software adds a step above whatever line is shaded in blue. For example, in Figure 8.23, if a step were to be added with the Collection Fractions step highlighted, the step would be added before this step. To add a step after Collection Fractions, have the End of Protocol step highlighted. 180 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series Figure 8.23. Programming the collection windows. Screenshot of the programmed windows. 2. Step 2: Turn on the lamp by clicking on the Lamp button on the left side of the screen and then select the “on”option and click OK (Figure 8.24). Figure 8.24. Programming Step 2: Lamp (UV Detector) Turn On. Chapter 8: Purification Protocol for BioLogic DuoFlow System 181 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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Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
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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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