Protein Expression and Purification Series - Bio-Rad

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CHAPTER 4 11 ml CULTURE PROTOCOL Protein Expression and Purification Series Centrifugation Purification Workflow Cell Culture and Induction Streaking starter plates to produce single bacterial colonies on agar plates. Pipet 10 µl BL21(DE3) E. coli containing pDHFR onto LB/Amp plate; streak out using sterile inoculation loop. Incubate overnight at 37°C. Overnight culture. Label the 50 ml conical containing 3 ml of sterile LB/Amp broth with your initials. Add 150 µl 20% glucose and single E. coli colony to 3 ml of LB/amp liquid media. Incubate overnight at 37°C in shaking incubator or incubator/tube roller. Prepare uninduced SDS-PAGE sample. Label 1.5 ml screwcap microcentrifuge tube Uninduced PAGE with your initials. Take 100 µl aliquot from overnight culture and prepare for SDS-PAGE per manual instructions. Measure cell density of overnight culture. Add 900 µl LB/amp to cuvette; take 100 µl aliquot from overnight culture and add to same cuvette. Using a spectrophotometer, measure absorbance at 600 nm (OD600). Prepare a subculture with a starting OD600 of . 0.3 Determine amount to add to LB/amp for subculture with a starting OD600 of 0.3. Remove LB/amp equivalent volume of calculated cells to be added from prewarmed 50 ml tube containing LB/amp. Add calculated amount of cells from overnight culture to prewarmed 50 ml tube. Measure OD600 of subculture and induce GST-DHFR-His expression. Incubate subculture for one hour at 37°C with shaking. Measure OD600 and record. Induce expression by adding 25 µl 100 mM IPTG to culture and incubate 4–24 hours at 37°C with shaking. 900 µl LB/amp + 150 µl 20% glucose Overnight culture + 25 µl IPTG 3 ml LB/amp 3 ml O/N culture (37°C O/N) (O/N at 37°C with shaking) 100 µl 100 µl LB/amp prewarmed to 37°C Prep Uninduced PAGE sample Measure OD600 (1 hr at 37°C with shaking) (4–24 hr at 37°C with shaking) 108 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series Centrifugation Purification Workflow Collecting cell pellet and lysing cells Prepare Induced SDS-PAGE sample. After induction is completed, take 100 µl aliquot from induced culture and prepare for SDS-PAGE per manual instructions. Pelleting induced cells. Label two 2 ml microcentrifuge tubes with Induced cell pellet and your team’s initials. Add 2 ml of induced culture into each of two 2 ml tubes. Centrifuge for two minutes at 16,000 x g. Carefully decant and discard the supernatant Repeat the addition of 2 ml induced culture to each tube; Centrifuge; decant; repeat once more until all cells are pelleted (three times total). Lysing induced cells. Add 250 µl Lysis buffer 1 to each of the two tubes containing a cell pellet. Using a vortexer or pipet, fully resuspend the pellets. Label a clean 2 ml microcentrifuge tube Lysed cells with your initials. Combine resuspended, lysed pellets into the lysed cells tube. Place lysed cells into dry ice/ethanol bath for five minutes. Remove and thaw completely. Repeat two more times. Add 500 µl Lysis buffer 2 to tube. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification + 250 µl Lysis buffer 1 into each tube 109 + 500 µl Lysis buffer 2 + 2 ml Induced Culture 100 µl + 2 ml Induced cell pellet Lysed cells freeze thaw Spin at 16,000 x g for 2 minutes Prep Induced PAGE sample Resuspend cell pellets; combine into 1 tube Perform lysis procedure X 3 X 3 CHAPTER 4 11 ml CULTURE 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 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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