Protein Expression and Purification Series - Bio-Rad

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CHAPTER 4 11 ml CULTURE PROTOCOL Protein Expression and Purification Series 7. Remove 50 µl of “ Insoluble fraction” and place it in a clean 1.5 ml screwcap microcentrifuge tube labeled “Insoluble PAGE” with your initals. Add 50 µl Laemmli sample buffer and mix thoroughly. 8. Heat the “ Soluble PAGE” and “Insoluble PAGE” samples at 95°C for five minutes. Note: Make sure that you are heating your blue SDS-PAGE samples and not your actual soluble and insoluble fractions! 9. Store the SDS-PAGE samples “ Soluble PAGE” and “Insoluble PAGE” at –20°C until ready to analyze via SDS-PAGE. 10. Store the soluble fraction and Insoluble fraction at –20°C until ready to purify the soluble fraction. 100 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series Affinity and Size Exclusion (Desalting) Purification of GST-DHFR-His Student Workstations Each student team requires the following items to purify their GST-DHFR-His samples using affinity chromatography followed by size exclusion chromatography (desalting): Material Needed for Each Workstation Quantity Soluble lysate fraction 600–750 µl Profinity IMAC Ni-charged resin slurry 250 µl slurry Distilled water 200 µl Equilibration buffer 500 µl Wash buffer 600 µl Elution buffer 400 µl Empty Micro Bio-Spin column with cap and yellow tip closure 1 Desalting column 1 Microcentrifuge tubes, 2 ml 6 Screwcap microcentrifuge tubes, 1.5 ml 4 Laemmli sample buffer (leftover from previous activity) 1 ml 2–20 µl adjustable-volume micropipet and tips 1 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 Tube roller or rocking platform 1 Water bath or dry bath set to 95°C 1 Microcentrifuge with variable speed setting > 16,000 x g 1 Pouring, washing, and equilibrating column 1. Snap off the bottom tab of the empty Micro Bio-Spin column, label the column with your initials and place the column in a clean 2 ml microcentrifuge tube. 2. 3. 4. Thoroughly resuspend the resin and pipet 200 µl of the slurry into the column. Centrifuge for 2 minutes at 1,000 x g to remove the packing. (See Appendix C—Instructions on how to calculate the speed that needs to be set to generate a relative centrifugal force, RCF, of 1,000 x g.) Discard the buffer that has collected in the 2 ml microcentrifuge tube. Place the column back into the 2 ml microcentrifuge tube. Note: The resin is provided as a 50% slurry in 20% ethanol. It is very important to thoroughly resuspend the resin before pipeting, or the incorrect quantity of resin will be aliquoted. Wash the column by gently adding 200 µl of distilled water to the top of the column taking care not to disturb the resin. Centrifuge for 2 minutes at 1,000 x g to remove the water. Discard the water that was collected in the microcentrifuge tube and place the column back into the 2 ml microcentrifuge tube. Equilibrate the column by gently adding 500 µl of Equilibration buffer and centrifuge for 2 minutes at 1,000 x g to remove the Equilibration buffer. Discard the buffer and the 2 ml microcentrifuge tube. Do not discard the column! Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 101 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 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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