Protein Expression and Purification Series - Bio-Rad

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Protein Expression and Purification Series Lab Session Centrifugation Purification continued Chapter Task Estimated Duration 7 4 and 8: Culturing, Expression, Lysis and SDS-PAGE Analysis for 11 ml Cultures and Purification Protocol for Centrifugation Process; and DHFR Analysis Optional stopping point: Store fractions at 4°C and SDS-PAGE samples at –20°C indefinitely Prepare desalting columns and desalt eluate fraction Prepare desalted eluate SDS- PAGE sample Optional stopping point: Store fractions at 4°C and SDS-PAGE samples at –20°C indefinitely Module Containing Materials 30 min Centrifugation Purification 15 min SDS-PAGE Electrophoresis Reheat SDS-PAGE samples 15–30 min N/A Electrophorese SDS-PAGE samples 0.5–1 hr SDS-PAGE Electrophoresis Rinse and stain gels 1.25+ hr* SDS-PAGE Electrophoresis Destain gels 8+ hr* N/A Measure and calculate desalted eluate concentration Prepare enzymatic assay reagents (Must be prepared within 3–4 hrs of use) 30 min N/A 15 min DHFR Enzymatic Assay Perform enzymatic analysis 2 hr Growth and Expression and DHFR Enzymatic Assay *Time indicated for these tasks is not hands-on time. It is the time needed for reactions to run, bacteria to grow, or time to incubate. 22 Timeline for the Laboratory Course: Centrifugation Purification
Protein Expression and Purification Series Centrifugation Purification Helpful Hints Checklist Overnight Cultures • 37°C incubation temperature of cultures is required. • If using a shaking incubator or shaking water bath, a rotational speed of 250–275 rpm is required. • If using a tube roller in an incubator, the tube roller should be in end over end mixing mode. Subcultures • Ensure the LB/amp medium is prewarmed to 37°C. • 37°C incubation temperature of cultures is required. • If using a shaking incubator or shaking water bath, a rotational speed of 250–275 rpm is requried. • If using a tube roller in an incubator, the tube roller should be in end over end mixing mode. Induction • Determine timeline for induction. Four hour induction provides optimal results. • 37°C incubation temperature of cultures is required. • If using a shaking incubator or shaking water bath, a rotational speed of 250–275 rpm is required. • If using a tube roller in an incubator, the tube roller should be in end over end mixing mode. Cell Lysis • Prepare lysozyme fresh (within 24 hours of use) for best results. • Make sure lysozyme was made in 2x PBS. • Make sure the cell pellets are completely resuspended in lysis buffer 1. Separating Soluble and Insoluble Fractions • The centrifuge must be able to generate 16,000 x g of force (RCF). RCF is not equal to RPM. Make sure that the correct conversion from RCF to RPM is calculated. For more information on this conversion please refer to Appendix C. Centrifugation Affinity Purification • The microcentrifuge must be able to generate 1,000 x g of force (RCF). RCF is not equal to RPM. Make sure that the correct conversion from RCF to RPM is calculated. For more information on this conversion please refer to Appendix C. • Do not use a mini-centrifuge for this procedure because they generally generate 2,000 x g (RCF), which is too much force for the affinity Ni-IMAC resin to withstand. • Ensure the Ni-IMAC resin is fully resuspended before preparing columns. • Ensure the sample binds to the resin for 20 minutes with rocking. Size Exclusion Purification (Desalting) • The microcentrifuge must be able to generate 1,000 x g of force (RCF). RCF is not equal to RPM. Make sure that the correct conversion from RCF to RPM is calculated. For more information on this conversion please refer to Appendix C. • Do not use a mini-centrifuge for this procedure because they generally generate 2,000 x g (RCF), which is too much force for the Bio-Gel P6 desalting resin to withstand. • Removal of the storage buffer from the desalting columns is important. Make sure to let the resin drain via gravity to remove the first portion of the storage buffer. • Make sure to use the 2 ml microcentrifuge tubes during the desalting process. 1.5 ml microcentrifuge tubes are not large enough to allow the buffer to flow from the column into the collection tube. • Verify that the desalted sample volume is not >150 µl (assuming two desalting runs using the same column). If the volume is >150 µl, then the column storage buffer was likely not removed properly. Timeline for the Laboratory Course: Centrifugation Purification 23
Page 1: Biotechnology Explorer TM Protein E
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Page 34 and 35: CHAPTER 1 BACKGROUND Protein Expres
Page 50 and 51: CHAPTER 2 FOCUS QUESTIONS Protein E
Page 56 and 57: 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 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX A TIPS AND HELPFUL HINTS P
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APPENDIX B RESULTS ANALYSIS Protein
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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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Protein Expression and Purification Series - Bio-Rad

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