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 Quantitation of Protein in Desalted Fraction Turn on spectrophotometer and set absorbance to 280 nm. Add 100 µl distilled water to clean UV compatible cuvette. Blank spectrophotometer with distilled water. Pipet 100 µl of your Desalted eluate sample (GST-DHFR-His) into clean UV compatible cuvette. Measure absorbance of sample at 280 nm. Return sample to 2 ml tube. Calculate concentration of GST-DHFR-His in your Desalted eluate fraction as described in instruction manual and record data. + 100 µl Distilled H 2 O Clean UV cuvette + 100 µl Desalted eluate Clean UV cuvette 116 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series Centrifugation Purification Workflow SDS-PAGE Electrophoresis to Check Expression and Purity Load, run, stain and destain the gel. Reheat SDS-PAGE samples at 95°C for two minutes. Centrifuge samples for two minutes at 16,000 x g. Using a fresh tip each time, load the following volumes of each sample into the appropriate well of your gel Lane Volume Sample 1 10 µl Precision Plus Dual Color standard 2 7.5 µl Uninduced PAGE 3 15 µl Induced PAGE 4 10 µl Insoluble PAGE 5 10 µl Soluble PAGE 6 10 µl Flowthrough PAGE 7 20 µl Wash PAGE 8 20 µl Eluate PAGE 9 20 µl Desalted eluate PAGE 10 10 µl Laemmli buffer Run the gel at 200 V for 30 minutes. (If using Mini-PROTEAN TGX precast gel, the gel can be run at 300 V for 15 minutes.) After the run is complete, remove the gel from the cassette and place it in the gel staining tray. Wash the gel three times with 50 ml of distilled water for five minutes each rinse. Discard all the rinse water. Stain the gel with 50 ml of Bio-Safe Coomassie stain for one hour. After one hour, discard the Bio-Safe Coomassie stain, add 100 ml of distilled water, and destain the gel overnight. Image the gel if you have an imaging system or dry the gel if you have a cellophane drying system. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 117 Heat 2 min at 95°C Spin 2 min at 16,000 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 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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