Protein Expression and Purification Series - Bio-Rad

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CHAPTER 3B ADVANCE PREP INSTRUMENTATION PROCES Protein Expression and Purification Series 4. BioLogic DuoFlow system ONLY: Note: The following instructions assume a basic working knowledge of use of the BioLogic DuoFlow system. If the instrument has not been run before, it is strongly suggested that a Starter kit (catalog #760-0135EDU) is run to familiarize yourself with the instrument components, nomenclature, and programming. a. Install a 5 ml sample loop on each instrument. Remove the 50 µl sample loop that was installed with the instrument and store in a safe place. Replace this sample loop with a 5 ml sample loop by attaching the fittings into port 6 and port 3 of the AVR7-3 injection valve. Please reference your DuoFlow system instruction manual (Section 4.2—Plumbing a DuoFlow System; Section 6—Plumbing the AVR7-3 Inject Valve) for more information. b. c. d. Injection port needle: It is critical that students use only the injection port needle that was supplied with the DuoFlow system instrument. This needle has a flat bottom (in contrast to the sharp, beveled edge on a traditional syringe needle). The injection port on the instrument has a filter that will be damaged if a syringe needle is used. Install column: Using fittings to convert the columns fittings from luer-style to ¼-28 style, attach the column after the AVR7-3 valve and before the UV detector. Make sure that the arrow on the column (if using a Bio-Scale mini cartridge) points in the correct direction of flow. Check flow rates of Pump A and Pump B: In manual mode, set the flow rate to 2.00 ml/min and place the pump inlet tubing for both Pump A and Pump B in high quality water. Set the AVR7-3 to Load. Place the waste outlet tubing coming from the SV3- 2 valve for the fraction collector into a graduated cylinder. Choose 100% A and start the pump. Allow Pump A to run for 5 minutes. Check the volume of water collected in the graduated cylinder. It should be close to 10 ml. Repeat for Pump B. If the volume is incorrect for either pump, purge the system to make sure there are no air bubbles present in the lines. Optional: Depending on teaching objectives, it is possible to program all of the instruments with the DHFR purification protocol beforehand, rather than have students perform this task. 5. Set up student workstations according to the student workstation list on pages 87-88. Quantitation of protein in desalted fractions 1. Turn on the spectrophotometer and allow it to warm up for at least 30 minutes. If using the Bio-Rad SmartSpec Plus spectrophotometer, step-by-step instructions are available in Appendix D for setting up your instrument and measuring the absorbance at 280 nm. If you are using another manufacturer’s spectrophotometer, please consult the instruction manual for that instrument for setting it up to read absorbance at 280 nm. Note: UV compatible cuvettes (trUView or quartz) capable of reading 50–100 µl samples must be used for this step. 2. Set up student workstations according to the student workstation list on pages 87-88. 86 Chapter 3B: Advance Preparation for Chromatography Instrumentation Protocols
Protein Expression and Purification Series Tasks to Perform After the Lab In order to ensure excellent, long-term performance of the BioLogic LP system or DuoFlow instrument, it is important to wash all tubing and components with high-quality water for short-term storage and with a 20% ethanol solution in water for long-term storage. A water wash will remove all salts that can precipitate and clog tubing and flow paths. If instruments are used regularly (at least once or twice a month) this wash should be sufficient. If instruments are used infrequently, washing with water followed by a 20% ethanol wash will wash out salts as well as provide some level of antibacterial activity with the ethanol. Make sure to wash out all tubing and all valve positions. Student Workstations 1. Advance preparation for handpacking a glass econo-column column with a flow adaptor If students are handpacking their own columns, the following workstations will need to be set up. Otherwise, if they are using prepacked cartridges this section can be skipped and proceed to section 2 below. Each student team requires the following items to handpack their own column: Material Needed for Each Workstation Quantity Glass Econo-Column column, 1 cm x 5 cm 1 Profinity IMAC Ni-charged resin slurry (50% slurry) 2 ml slurry Flow adaptor for 1 cm ID columns 1 2-way stopcock 1 10 ml syringe 1 Beaker filled with 200 ml of distilled water 1 50 ml beaker for waste 1 100-1,000 µl adjustable-volume micropipet and tips 1 Marking pen 1 2. Affinity purification of GST-DHFR-His protein Each student team requires the following items to purify their GST-DHFR-His samples using affinity chromatography: Material Needed for Each Workstation Quantity Soluble lysate fraction 5–7.50 ml BioScale Mini Profinity Ni-IMAC cartridge 1 A glass Econo-Column column handpacked with 1 ml of Profinity IMAC Ni-Charged resin 1 10 ml syringe 1 Distilled water 500 ml Buffer A 70 ml Buffer B 40 ml Collection tubes for fraction collector 30 Marking pen 1 Common Workstation Quantity BioLogic system instrumentation with sample loop installed 1–4 Injection needle (DUOFLOW System ONLY) 1–4 Chapter 3B: Advance Preparation for Chromatography Instrumentation Protocols 87 CHAPTER 3B ADVANCE PREP INSTRUMENTATION PROCES
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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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Page 50 and 51: CHAPTER 2 FOCUS QUESTIONS Protein E
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CHAPTER 6 HANDPACKING COLUMNs Prote
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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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