Protein Expression and Purification Series - Bio-Rad

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APPENDIX B RESULTS ANALYSIS Protein Expression and Purification Series 250 kD 150 100 75 50 37 25 20 15 10 Flowthrough Wash Eluate 1 2 3 4 5 6 7 8 9 10 imidazole absorbance Figure 6. BioLogic DuoFlow system chromatogram showing a large peak of flowthrough (between 2–10 minutes) and the smaller peak of purified GST-DHFR-His eluate (between 14–15 minute). The elevated baseline absorbance between 15–20 minutes represents the increased absorbance from the imidazole in the elution buffer. Step 3: Check that purification worked by analysis of the chromatogram. Results: There should be a large peak around 2–8 minutes with a high absorbance at 280 nm that is from the flowthrough of proteins that did not bind to the Ni-IMAC column. There should also be a defined peak around 14–16 minutes that contains the GST-DHFR-His that was eluted with the addition of 250 mM imidazole from the Ni-IMAC column. Figure 7. Lane 1, molecular weight markers; lane 2, soluble fraction; lane 3, flowthrough; lane 4, wash; lane 5, fraction A; lane 6, desalted fraction A; lane 7, fraction B; lane 8, desalted fraction B; lane 9, fraction C; lane 10, desalted fraction C. GST-DHFR-His lysozyme Step 4: Check that the GST-DHFR-His is present in the fractions chosen after determining delay volume. There should be a band of approximately 43 kD that accounts for the majority of the protein in lane(s) 5, 7, and/or 9 at approximately 43 kD (between the 37 kD and 50 kD molecular weight markers). The purity and darkness of the GST-DHFR-His band(s) will depend on the specific culture and induction times used. Step 5: Check that desalting of GST-DHFR-His was successful. There should be a band of approximately 43 kD that accounts for the majority of the protein in lane(s) 6, 8 and/or 10 that are of comparable size to the main band(s) in lanes 5, 7, and/or 9 and about the same darkness. The absorbance value of this fraction at 280 nm should give a significant value above the blank absorbance. 216 Appendix B: Purification Results Analysis
Protein Expression and Purification Series Step 6: Check that the purified GST-DHFR-His has enzymatic activity. Results: The “No Substrate Control Reaction” should show a fairly flat line with an absorbance around 0.2–0.4 (Fgure 8). The enzyme reaction should show a decrease in absorbance at 340 nm over the 150 seconds of reading time (Figure 9). The calculated reaction rate will vary depending on the amount of GST-DHFR-His purified. Absorbance at 340 nm Figure 8. No substrate control enzymatic activity reaction. Absorbance at 340 nm Figure 9. GST-DHFR-His enzyme activity reaction. Appendix B: Purification Results Analysis No Substrate Control Reaction Time in seconds GST-DHFR-His Enzyme Reaction Time in seconds 217 APPENDIX B RESULTS ANALYSIS
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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 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 210 and 211: APPENDIX A TIPS AND HELPFUL HINTS P
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Page 214 and 215: APPENDIX B RESULTS ANALYSIS Protein
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
Page 226 and 227: APPENDIX F BIOLOGIC LP SYSTEM SETUP
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
Page 232 and 233: APPENDIX G BIOMANUFACTURING Protein
Page 234 and 235: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 242 and 243: APPENDIX I GLOSSARY Protein Express
Page 244 and 245: APPENDIX I GLOSSARY Protein Express
Page 246 and 247: APPENDIX J REFERENCES, LEGAL NOTES
Page 250: Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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