Protein Expression and Purification Series - Bio-Rad

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CHAPTER 5 100 ml CULTURE PROTOCOL Protein Expression and Purification Series Collecting cell pellet and lysing cells Student Workstations Each student team requires the following items to isolate their cell pellet, to prepare an induced cell sample for SDS-PAGE analysis, and to lyse their cells: Material Needed for Each Workstation Quantity Induced cell culture 100 ml Lysis buffer 5 ml Equilibration buffer 5 ml 250 ml centrifuge bottles capable of withstanding 4,500 x g 1 50 ml sterile conical tube 1 Screwcap microcentrifuge tube, 1.5 ml 1 Laemmli sample buffer (left over from previous activity) 1 ml 20–200 µl adjustable-volume micropipet and tips 1 100–1,000 µl adjustable-volume micropipet and tips 1 Marking pen 1 Common Workstation Quantity Water bath or dry bath set to 95°C 1 Microcentrifuge with variable speed setting >16,000 x g 1 Benchtop or floor centrifuge with rotor for 250 ml centrifuge bottles 1 Vortexer 1 Dry ice/ethanol bath 1 Prepare induced SDS-PAGE sample 1. Label a 1.5 ml screwcap microcentrifuge tube “ Induced PAGE” with your initials. 2. 3. 4. 5. 6. Aliquot 100 µl of the induced culture into the tube. Centrifuge the tube at 16,000 x g for 2 minutes. Use a pipet to gently remove and discard the supernatant without disturbing the pellet. Add 100 µl Laemmli sample buffer to the pellet and fully resuspend the pellet by pipeting up and down or vortexing. Heat the tube at 95°C for five minutes. 7. Store the sample at –20°C until ready to analyze via SDS-PAGE. Pelleting induced cells 1. Place the 100 ml of induced culture in a 250 ml centrifuge bottle that will fit into your benchtop or floor model centrifuge. 2. Make sure that your vessel is balanced with either another student’s bottle or with a bottle filled with water. If you are balancing your bottle with another student’s bottle, sterile water can be added to achieve balanced bottles. 124 Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification
Protein Expression and Purification Series 3. 4. Centrifuge the cell pellet at 4,500 x g for 10 minutes. When the centrifugation is done the supernatant should look transparent. If it is not transparent centrifuge the bottle for another 10 minutes. Carefully decant or pipet the supernatant making sure not to disturb the pellet and to dispose of the supernatant properly. Store the cell pellet at –20°C or continue on to lysing induced cells. Lysing induced cells 1. Add 5 ml of lysis buffer to the cell pellet and using a pipet or a vortexer fully resuspend the cells in the lysis buffer. Make sure that there are no cell clumps remaining in the vessel. Pour the resuspended cells in lysis buffer into a 50 ml conical tube and label it “Lysed Cells” with your initials. Notes: As lysis occurs, the solution will get more viscous. Vigorous vortexing and pipeting might be necessary to resuspend the pellet. Use lab tap for the labeling of the 50 ml conical tube since ethanol in the dry ice/ethanol bath will remove the markings from the tube, but not from tape. 2. 3. 4. Place the tube of lysed cells in the dry ice/ethanol bath for five minutes. After five minutes remove the tube from the dry ice/ethanol bath and thaw completely. It is acceptable to thaw in a 37°C waterbath. Do not thaw by hand to avoid freezer burn to your hand. Note: Be careful not to allow the dry ice/ethanol to come in contact with your skin. Repeat two more freeze–thaw steps for a total of three freeze–thaw cycles. After the final thaw step add 5 ml Equilibration buffer and thoroughly mix by pipetting or vortexing. 5. The lysed cells can be stored at –20°C until they are ready to be purified. Note: Another method that can be used for efficient lysis is freezing the cells in a –70°C freezer. The cells can be frozen at –70°C overnight and then thawed completely when separation of the soluble from the insoluble fraction is to be performed. If neither dry ice/ethanol or a –70°C freezer is availalble, it is possible to perform two freeze/thaw cycles using a –20°C freezer. Samples can be placed at –20°C overnight (check that samples are completely frozen), thawed the next day and then placed at –20°C overnight again before purification. Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification 125 CHAPTER 5 100 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 3B ADVANCE PREP INSTRUMENTA
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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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