Protein Expression and Purification Series - Bio-Rad

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CHAPTER 9 DHFR ENZYMATIC ASSAY Protein Expression and Purification Series Solving for ΔC (mol/liter/min) = ΔOD ε x l Example: For the data from the graphs and calculations above: Solving for ΔC (mol/liter/min) = 0.025/mi = 4 x 10 -6 mol/liter/min 6220 M -1 cm -1 x 1 cm Calculate how many moles of NADPH your GST-DHFR-His sample can break down per minute and record below. ΔC (mol/liter/min) = ΔOD (min -1 ) = __________________(mol/liter/min) 6220 M -1 cm -1 x 1 cm 208 Chapter 9: DHFR Enzymatic Activity Assay Student Protocol
Protein Expression and Purification Series DHFR Enzymatic Activity Assay Determining which fraction to test for enzyme activity. Review your SDS-PAGE gel from your purification to determine which fraction(s) to test. Note that centrifugation purification will have one desalted fraction while instrumentation-based purification will have three fractions. Set up spectrophotometer for kinetics measurements at 340 nm. Blanking the instrument. Add 985 µl 1x PBS to cuvette; place in instrument, read as blank. Save cuvette with PBS. Running the no substrate control reaction. Add 6 µl of 10 mM NADPH to cuvette containing 985 µl 1x PBS. Add 15 µl of purified, GST-DHFR-His eluate to cuvette. Cover cuvette with parafilm and invert 10 times to mix Place cuvette in spectrophotometer and begin kinetics run. As run is proceeding, record absorbance value every 15 seconds for 150 seconds. Remove and save cuvette from the spectrophotometer. Running the enzymatic reaction with the GST-DHFR-His, NADPH (cofactor) and DHF (substrate). Note: The enzyme reaction should be prepared while standing at the spectrophotometer. The reaction occurs extremely quickly and it is necessary to place the cuvette in the spectrophotometer and start the readings as quickly as possible once the DHF has been added. Add 5 µl of 10 mM DHF to the cuvette already containing 1x PBS, your GST-DHFR-His sample and NADPH. Quickly cover the cuvette with parafilm and invert five times. Place the cuvette in the spectrophotometer and begin kinetics run. As run is proceeding, record absorbance value every 15 seconds for 150 seconds. Remove cuvette from the spectrophotometer. Calculate the activity following the instructions in the manual. Chapter 9: DHFR Enzymatic Activity Assay Student Protocol 209 + 985 µl 1x PBS + 15 µl GST-DHFR-His eluate 1x PBS 1x PBS NADPH GST-DHFR-His eluate 1x PBS NADPH GST-DHFR-His eluate + 6 µl 10 mM NADPH + 5 µl 10 mM DHF CHAPTER 9 DHFR ENZYMATIC ASSAY
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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 158 and 159: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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Page 210 and 211: APPENDIX A TIPS AND HELPFUL HINTS P
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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
Page 222 and 223: APPENDIX D SETTING UP THE SMARTSPEC
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
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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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