Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Purification Workflow for BioLogic DuoFlow System Affinity Purification of GST-DHFR-His Protein Follow the preceding protocols for your instrument and either handpacked columns or prepacked cartridge purificaton. Post-purification: Choosing your flowthrough, wash, and three eluted GST-DHFR-His fractions Examine your chromatogram and determine the fractions that you feel are most likely to contain your flowthrough and wash, and three fractions which might contain your eluted GST-DHFR-His. Remember that you need to calculate the delay volume to help determine which fractions contain your samples. Record the five fraction numbers you will examine at right. Label 1.5 ml screwcap microcentrifuge tubes according to the fraction as indicated on right. Add 50 µl of fraction to 50 µl Laemmli buffer into corresponding 2 ml screwcap tube. Mix well. Heat all samples at 95°C for five minutes. BioLogic DuoFlow System Flowthrough fraction: Wash fraction: First eluted GST-DHFR-His fraction: Second eluted GST-DHFR-His fraction: Third eluted GST-DHFR-His fraction: Preparing SDS-PAGE samples of flowthrough, wash and eluted GST-DHFR-His fractions Flowthrough PAGE + 50 µl fraction Wash PAGE SDS-PAGE sample Fraction A PAGE Heat 2 min at 95°C Fraction B PAGE + 50 µl Laemmli Buffer Fraction C PAGE 196 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series Purification Workflow for BioLogic DuoFlow 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 two 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 minutew 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 ~150ul of desalted GST-DHFR-His for each elution fraction. Chapter 8: Purification Protocol for BioLogic DuoFlow System 197 Desalted A, B or C 2 min at 1,000 x g + 75 µl eluate 4 min at 1,000 x g X 2 CHAPTER 8 BIOLOGIC DUOFLOW 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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CHAPTER 5 100 ml CULTURE PROTOCOL P
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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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Page 146 and 147: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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Page 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
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Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
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APPENDIX J REFERENCES, LEGAL NOTES
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Life Science Group Bulletin 1665067
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