Protein Expression and Purification Series - Bio-Rad

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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOCOL Protein Expression and Purification Series Purification Workflow for BioLogic LP System Desalting your three eluted GST-DHFR-His fractions Label three Micro Bio-Spin desalting columns A, B, and C. These fractions correspond to the three fractions you chose from your chromatogram. Prepare desalting columns by inverting sharply several times to resuspend gel. Snap off bottom tabs and place each column into a 2 ml microcentrifuge tube. Remove green top cap. If the column does not begin to flow, push the cap back on the column and then remove it again to start the flow. Allow excess packing buffer to drain by gravity to top of resin bed. After draining, place columns in clean 2 ml tubes. Centrifuge for 2 minutes at 1,000 x g. Discard remaining packing buffer and collection tubes. Label three clean 2 ml microcentrifuge tubes Desalted A, Desalted B, Desalted C. Carefully apply 75 µl of each of your eluted fractions directly to the center of the corresponding column. Be careful not to touch resin with pipet tip. Centrifuge for four minutes at 1,000 x g. Carefully apply another 75 µl of each of your eluted fractions to the corresponding column and centrifuge again. After second spin, discard columns. You will have ~150 µl of Desalted GST-DHFR-His for each elution fraction. Desalted A, B or C 2 min at 1,000 x g + 75 µl eluate 4 min at 1,000 x g X 2 162 Chapter 7: Purification Protocol for BioLogic LP System
Protein Expression and Purification Series Purification Workflow for BioLogic LP System Preparing SDS-PAGE Samples of Desalted GST-DHFR-His Fractions Prepare SDS-PAGE samples. Add 25 µl of each fraction to 25 µl Laemmli buffer in 2 ml screwcap tube. Mix well. Heat all samples at 95°C for five minutes. Chapter 7: Purification Protocol for BioLogic LP System 163 + 25 µl fraction Desalted A PAGE SDS-PAGE sample Desalted B PAGE Heat 2 min at 95°C + 25 µl Laemmli Buffer Desalted C PAGE CHAPTER 7 BIOLOGIC LP SYSTEM 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 3B ADVANCE PREP INSTRUMENTA
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CHAPTER 4 11 ml CULTURE PROTOCOL Pr
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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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