Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series 3. 4. Choose Isocratic Flow from the left-side menu. Change the composition of the 2x PBS (Buffer A) to 98%. The % Composition for Buffer B will automatically change to 2%. Change the volume to 5.00 ml and the flow rate to 2.00 ml/min, and then press OK (Figure 8.25). Figure 8.25. Programming Step 3: Equilibrating the column with 2x PBS + 5 mM imidazole for 5 CV at 2 ml/min. Continue programming steps 4–9 as defined in Table 8.2 below. Your final protocol should look like Figure 8.26. Table 8.2. Programming the Protocol Step Number Start (ml) Purpose Programming 1 0.00 Programming fraction collector 2 0.00 Turn the lamp on (if not already on) Collect 4.00 ml fractions during the sample injection and wash steps and then 1.00 ml fraction during the elution step Lamp Choose on 3 0.00 Equilibrate column Isocratic flow with 98% 2x PBS and 2% 2x PBS, 250 mM Imidazole for 5 ml at 2.00 ml/min 4 5.00 Zero baseline Zero baseline to set UV baseline to 0.0 5 5.00 Inject sample onto the column Load Inject sample, static loop: Inject 6.0 ml* sample at 1.00 ml/min. The injection occurs using 98% 2x PBS and 2% 2x PBS, 250 mM Imidazole 6 11.00 Wash the column Isocratic flow with 96% 2x PBS and 4% 2x PBS + 250 mM Imidazole for 11 ml at 2.00 ml/min 7 22.00 Elute GST-DHFR-His Isocratic flow with 0% 2x PBS and 100% 2x PBS, 250 mM Imidazole, for 8 ml at 2.00 ml/min 8 30.00 Final column wash Isocratic flow with 100% 2x PBS and 0% 2x PBS, 250 mM Imidazole for 15 ml at 2.00 ml/min 9 45.00 Turn off lamp Lamp Choose off 45.00 End of protocol *A slightly larger volume (6 ml) than the sample loop (5 ml) is used to ensure all the sample is fully pushed onto the column. 182 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series Figure 8.26. Protocol for purifying GST-DHFR-His. Chapter 8: Purification Protocol for BioLogic DuoFlow System 183 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 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
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Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
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
Page 226 and 227: APPENDIX F BIOLOGIC LP SYSTEM SETUP
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Page 230 and 231: 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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