Protein Expression and Purification Series - Bio-Rad

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CHAPTER 3A ADVANCE PREP CENTRIFUGATION PROCES Protein Expression and Purification Series Advance Preparation for Cell Lysis Laboratory In this Cell Lysis activity, cells that have been expressing GST-DHFR-His will be pelleted and a lysis solution will be added. Lysis will be accomplished by multiple freeze-thaw steps using a dry ice/ethanol bath. The lysate will then be centrifuged to separate the soluble components from insoluble components. SDS-PAGE samples will be prepared for the insoluble and soluble cell lysate fractions. Cell Lysis Laboratory Checklist Components from Protein Expression and Purification Series Where Provided (✔) Lysozyme Growth and Expression Module ❒ Sterile water Growth and Expression Module ❒ Imidazole stock solution Growth and Expression Module ❒ 10x PBS Growth and Expression Module ❒ Laemmli sample buffer SDS-PAGE Electrophoresis Module ❒ Microcentrifuge tubes, 2 ml Growth and Expression Module ❒ Screwcap microcentrifuge tubes, 1.5 ml Growth and Expression Module ❒ Required Accessories (Not Provided) Quantity (✔) Dry ice 1 block ❒ Ethanol 0.5–2 L ❒ Microcentrifuge with variable speed setting >16,000 x g 1 ❒ 20–200 µl adjustable-volume micropipets and tips 12 ❒ 100–1,000 µl adjustable-volume micropipets and tips 12 ❒ Marking pens 12 ❒ 10 ml syringes 12 ❒ 22 gauge syringe needles 12 ❒ Water bath or dry bath set to 95°C 1 ❒ Tasks to Perform Prior to the Lab Collecting cell pellet and lysing cells 1. Prepare 2x PBS: To prepare 20 ml of 2x PBS, combine 4 ml of 10x PBS and 16 ml of distilled water and mix thoroughly. 2x PBS can be stored at room temperature and is good for one year. The 2x PBS is 20 mM sodium phosphate and 300 mM NaCl. 2. Prepare lysozyme: No more than one day in advance, rehydrate lysozyme by adding 500 µl of sterile water to the 25 mg of lyophilized lysozyme to generate a 50 mg/ml solution. Mix gently to aid in the resuspension. Keep the vial of lysozyme on ice or in a refrigerator until use. Once resuspended, it should be used within 24 hours. 3. Preparation of Lysis buffer 1: To prepare 20 ml of Lysis buffer 1, combine 20 ml of 2x PBS with 200 µl 50 mg/ml lysozyme solution. Store the Lysis buffer 1 at 4°C until ready to use. Once made, it should be used within 24 hours. The Lysis buffer 1 is 20 mM sodium phosphate, 300 mM NaCl and 0.5 mg/ml lysozyme. Aliquot 500 µl of Lysis buffer 1 into a 2 ml microcentrifuge tube for each workstation. 62 Chapter 3A: Advance Preparation for Centrifugation Protocols
Protein Expression and Purification Series 4. Prepare Lysis buffer 2/Wash buffer: To prepare 50 ml of Lysis buffer 2/Wash buffer, combine 38 ml of distilled water, 10 ml of 10x PBS and 2 ml of Imidazole stock solution and mix thoroughly. Lysis buffer 2/Wash buffer should be stored at 4°C and is good for one month. The Lysis buffer 2/ Wash buffer is 20 mM sodium phosphate, 300 mM NaCl and 10 mM imidazole. Aliquot 500 µl of Lysis buffer 2/Wash buffer into a 2 ml microcentrifuge tube for each workstation. 5. Prepare a dry ice/ethanol bath: Right before the lab period, add ethanol to a beaker. Add dry ice to the beaker until it no longer melts completely and pieces can still be seen in the solution. 6. Set up student workstations according to the student workstation list shown below. Separating soluble from insoluble induced cell fractions and prepare SDS-PAGE samples 1. Thaw lysates: If the cell lysates have been stored in a –20°C freezer, take them out at least one hour before the laboratory period and allow the lysates to thaw at room temperature. If the lysates do not thaw within one hour, they can be briefly warmed to 37°C to thaw completely. 2. Set up student workstations according to the student workstation list shown below. Student Workstations Each student team requires the following items to 1) collect their cell pellet, 2) lyse their cells, 3) separate the soluble from the insoluble lysate fraction, and 4) prepare samples for SDS-PAGE analysis: 1. Collecting cell pellet and lysing cells Material Needed for Each Workstation Quantity Induced cell culture 1 ml Lysis buffer 1 500 µl Lysis buffer 2/Wash buffer 500 µl Microcentrifuge tubes, 2 ml 3 Screwcap microcentrifuge tube, 1.5 ml 1 Laemmli sample buffer (leftover 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 Dry ice/ethanol bath 1 Vortexer 1 Chapter 3A: Advance Preparation for Centrifugation Protocols 63 CHAPTER 3A ADVANCE PREP CENTRIFUGATION PROCES
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Biotechnology Explorer TM Protein E
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Protein Expression and Purification
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Page 12 and 13: Protein Expression and Purification
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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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