Protein Expression and Purification Series - Bio-Rad

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Protein Expression and Purification Series Optional Materials Centrifugation BioLogic LP/BioLogic Purification DuoFlow Purification Autoclave 1 1 Vortexer (catalog #166-0610EDU) 1 N/A Imaging system (catalog# 170-8270EDU with 170-8272EDU) 1 1 Refills Available Separately Each individual module is available to order as a stand alone. In addition, certain refill items are also available: Growth and Expression reagent pack, catalog #166-5057EDU, includes ampicillin (60 mg), LB agar pouch (20 g), LB capsules (12), lysozyme (25 mg), BL21(DE)3 with pDHFR, 1M IPTG (0.1 ml), sterile water (2.5 ml), 10x PBS (100 ml), imidazole stock solution (200 ml), and 20% sterile glucose solution (4 ml) LB nutrient agar powder, 20 g, catalog #166-0600EDU LB nutrient agar powder, 500 g, catalog #166-0472EDU Ampicillin, 30 mg, catalog #166-0407EDU 10x PBS, 100 ml, catalog #166-2403EDU Sterile water, 500 ml, catalog #163-2091EDU Petri dishes, 60 mm, sterile, 500, catalog #166-0470EDU Inoculation loops, 10 µl, sterile, 80, catalog #166-0471EDU EZ Micro test tube, 2 ml, 500/box, catalog #223-9430EDU 1.5 ml conical tubes, with separate O-ring screwcaps, 500, catalog #224-0100EDU 1.5 ml conical tubes, with installed O-ring screwcaps, sterilized, 500, catalog #224-0110EDU 10x TGS buffer, 1 L, catalog #161-0732EDU Laemmli sample buffer, 30 ml, catalog #161-0737EDU Bio-Safe Coomassie stain, 1 L, catalog #161-0786EDU Precision Plus Protein Dual Color standards, catalog #161-0374EDU Jellyfish foam floating racks, 8 racks, catalog #166-0479EDU Gel staining trays, 4, catalog #166-0477EDU Profinity IMAC Ni-charged resin, 10 ml, catalog #156-0131EDU 1.0 cm x 5 cm, 2-pk Glass Econo-Column columns, catalog #737-1007EDU Micro Bio-Spin 6, Tris 25/pkg, catalog # 732-6221EDU Bio-Scale Mini cartridge, IMAC, 5 x 1 ml, catalog #732-4610EDU Flow adaptor, 1.0 cm Column ID, catalog #738-0014EDU 14 Materials Required But Not Supplied
Protein Expression and Purification Series Course Objectives The Protein Expression and Purification Series is appropriate for the laboratory portion of an undergraduate (or early graduate) course in Biotechnology, Biochemistry, Molecular Biology, Cell Biology, Recombinant DNA Techniques, or advanced high school biotechnology related courses. It would also be suitable for students doing independent research. It would be excellent for inclusion in biotechnology degree programs offered by community or technical colleges. The exercise could also prove useful for employers in the biotechnology, pharmaceutical, or industrial sectors as an introduction or refresher to protein purification techniques, and particularly as an introduction to using chromatographic instrumentation. Due to recent advances in the area of protein technology, the actual laboratory procedures are routine, safe and relatively inexpensive, provided basic laboratory equipment is available. Measures have been taken to ensure the safety of the reagents used. While proper laboratory safety techniques must always be used, the reagents provided are safe to use in the classroom. In order to complete the laboratory project in five to eight sessions, it is assumed that students meet at least once per week in a three-hour laboratory session, and that students can meet during the week to carry out a quick laboratory task or two. Specific Objectives Met by This Project 1. Students will experience a wide range of laboratory techniques. Some of the techniques implemented in this protein-based project are: cell culturing techniques, protein expression and purification, basic micropipetting, gel electrophoresis, and enzymatic analyses. 2. Students will see that these individual techniques are just steps in a longer investigatory process. Few researchers can complete an entire research project in one or two 3-hour laboratory sessions (the timeframe of most commercially available kits), so this five to eight period project more accurately reflects what goes on in a contemporary molecular biology laboratory. 3. Students will be active participants in the process. There are numerous occasions during the project when students are asked to troubleshoot their results, or to make judgments about what to do next. This exercise does not take a simple “cookbook” approach, but rather it involves more critical thinking. Course Objectives 15
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Page 34 and 35: CHAPTER 1 BACKGROUND Protein Expres
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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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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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