Protein Expression and Purification Series - Bio-Rad

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CHAPTER 4 11 ml CULTURE PROTOCOL Protein Expression and Purification Series Centrifugation Purification Workflow Separating Soluble from Insoluble Induced Cell Fractions and Preparation of SDS-PAGE Samples Separate soluble from insoluble cell lysate components. Centrifuge for 20 minutes at 16,000 x g (thaw first if necessary). Label 2 ml microcentrifuge tube Soluble fraction with your initials. The supernatant contains the soluble fraction, and the pellet contains the insoluble fraction. Gently pour the supernatant into the tube labeled soluble fraction. Relabel the Lysed cells tube Insoluble fraction. Add 1 ml Lysis buffer 2 to the insoluble fraction tube. The insoluble pellet contains a large quantity of genomic DNA, making it viscous and difficult to resuspend. Using a syringe with 22 gauge needle, repeatedly pull the solution through the syringe to shear the pellet and decrease the viscosity. Label two clean 1.5 ml screwcap microcentrifuge tubes Soluble PAGE and Insoluble PAGE. Take a 50 µl aliquot from each fraction Soluble fraction and Insoluble fraction and prepare for SDS-PAGE per manual instructions Store the SDS-PAGE samples, Soluble and Insoluble fractions at –20°C until ready for further steps. Lysed cells (thawed) Soluble Fraction Insoluble Fraction Insoluble Fraction 50 µl Prep Insoluble PAGE sample Spin 20 min at 16,000 x g Decant supernatant into tube labeled Soluble raction + 1 ml Lysis buffer 2 Shear insoluble fraction with syringe needle to resuspend pellet Soluble Fraction 50 µl Prep Soluble PAGE sample 110 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series Centrifugation Purification Workflow Affinity and Size Exclusion (Desalting) Purification of GST-DHFR-His Pouring, washing and equilibrating column. Snap off bottom tab of empty Micro Bio-Spin column, label the column with your initials, remove cap and place in 2 ml microcentrifuge tube. Add 200 µl of IMAC resin slurry to empty column. Centrifuge for two minutes at 1,000 x g. After spin, discard buffer that has collected in the 2 ml microcentrifuge tube. Add 200 µl of distilled water to column. Centrifuge for two minutes at 1,000 x g. After spin, discard water from 2 ml microcentrifuge tube. Add 500 µl of Equilibration buffer to column. Centrifuge for two minutes at 1,000 x g. After spin, discard Equilibration buffer and 2 ml microcentrifuge tube. The column is now ready to use. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 111 Snap off bottom tab and remove cap + 200 µl IMAC resin slurry Spin 2 min at 1,000 x g + 200 µl dH 2 O Spin 2 min at 1,000 x g + 500 µl Equilibration buffer Spin 2 min at 1,000 x g 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 2 FOCUS QUESTIONS Protein E
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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CHAPTER 3A ADVANCE PREP CENTRIFUGAT
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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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