Protein Expression and Purification Series - Bio-Rad

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APPENDIX B RESULTS ANALYSIS Protein Expression and Purification Series Step 5: Check that desalting of GST-DHFR-His was successful. Lane 9 is the desalted GST-DHFR-His eluate fraction. Results: There should be a band of approximately 43 kD that accounts for the majority of the protein in lane 9 that is of comparable size to the main band in lane 8 and about the same darkness. The absorbance value of this fraction at 280 nm should give a significant value above the blank absorbance. 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 (Figure 2, below). The enzyme reaction should show a decrease in absorbance at 340 nm over the 150 seconds of reading time (Figure 3, below). The calculated reaction rate will vary depending on the amount of GST-DHFR-His purified. Absorbance at 340nm Figure 2. No substrate control enzymatic activity reaction. Absorbance at 340nm Figure 3. GST-DHFR-His enzyme activity reaction. No Substrate Control Reaction Time in seconds GST-DHFR-His Enzyme Reaction Time in seconds 214 Appendix B: Purification Results Analysis
Protein Expression and Purification Series BioLogic LP System and BioLogic Duoflow System Purification Results Analysis The gel image below in Figure 4 shows how typical results using the Chromatography Instrumentation Purification Process should appear. Listed below are common steps that can help you analyze your results. 1 2 3 4 5 Figure 4. Lane 1, molecular weight markers; lane 2, uninduced sample; lane 250 kD 3, induced sample; lane 4, insoluble fraction; lane 5, soluble fraction. 150 100 75 50 37 25 20 15 10 Step 1: Check that induction occurred. The GST-DHFR-His protein runs on SDS-PAGE gels at an apparent MW of approximately 43 kD. (Note that the protein runs at this weight despite the actual protein MW of approximately 52 kD.) Results: An obvious band should appear in lane 3 that is not present in lane 2 between the 37kD and 52 kD molecular weight markers if the GST-DHFR-His has been strongly induced. Step 2: Check that lysis worked. Lanes 4 and 5 represent the insoluble and soluble fractions, respectively. Results: If the GST-DHFR-His was strongly induced and the cells were lysed, a band should be present in lane 5 that is approximately 43 kD. It should be the same size as the band that occurred in lane 3 (falls between the 37kD and 50 kD molecular weight markers). Please note that the large band that is apparent at approximately 12 kD in lanes 4 and 5 is the lysozyme used to lyse the cells and not GST-DHFR-His. GST-DHFR-His is expressed in both a soluble and insoluble form. It is likely that there will be strong bands in both lanes 4 and 5 at around 43 kD, representing both insoluble and soluble GST-DHFR-His respectively. As long as there is a strong band in lane 5, lysis has occurred, and soluble GST-DHFR-His has been released. Figure 5. BioLogic LP Chromatogram showing a large peak of flowthrough (between 2–8 minutes in the chromatogram above) and the smaller peak of purified GST-DHFR-His eluate (between 14–16 minutes in the chromatogram above). Appendix B: Purification Results Analysis Induced GST-DHFR-His lysozyme Flowthrough Wash Eluate 215 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 164 and 165: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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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 216 and 217: 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
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
Page 228 and 229: APPENDIX F BIOLOGIC LP SYSTEM SETUP
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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