Protein Expression and Purification Series - Bio-Rad

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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOCOL Protein Expression and Purification Series 250 150 100 75 50 37 25 20 15 10 Flowthrough Wash Elute Column wash 1 2 3 4 5 6 7 8 9 10 Figure 7.16. Example of fractions selected from the chromatogram and the corresponding SDS-PAGE results. Lane 1. Precision Plus Dual Color standards Lane 2. Soluble fraction of E. coli cell lysate.. A band is present that is the GST-DHFR-His which is soluble. The large band at approximately 12 kDa is the lysozyme, which is also soluble, that was used to lyse the cells open. Lane 3. Flowthrough fraction that did not bind to the Ni-IMAC resin. This is the fraction of proteins from the soluble fraction that did not bind to the Ni-IMAC resin. The soluble fraction was suspended in 20 mM sodium phosphate buffer, 300 mM NaCl and 5 mM imidazole. The high salt helped prevent E. coli proteins and the lysozyme from non-specifically sticking to the Ni-IMAC beads. The 5 mM imidazole helped prevent E. coli proteins with multiple histidine groups from binding to the Ni-IMAC beads. There is a decrease in the amount of GST-DHFR-His in the flowthrough fraction versus the soluble fraction (lane 2) and this is representative of the GST-DHFR-His binding to the Ni-IMAC resin. Lane 4. Wash fraction. This fraction contains proteins that were washed off the Ni-IMAC beads when a wash buffer that has a slightly higher imidazole level (10 mM) was added to wash off more non-specifically bound proteins. No GST-DHFR-His should wash off in this fraction since 10 mM imidazole is not enough to compete with the 6 histidine tag of GST-DHFR-His bound to the Ni-IMAC resin. Lanes 5, 7, and 9. Eluate fraction of GST-DHFR-His. These fractions contain the GST-DHFR-His. The elution buffer has 250 mM imidazole in it and this level of imidazole competes with the six histines of GST-DHFR-His and knocks them off the Ni sites and hence the GST-DHFR-His elutes or comes off the resin to be collected. These fractions are predominantly GST-DHFR-His protein relative to the unpurified soluble fraction in lane 2 that contains many other proteins. Lane 6, 8, and 10. Desalted GST-DHFR-His. These fractions contain the purified GST-DHFR-His but have had the 250 mM imidazole removed. Also, since the desalting column removes smaller molecular weight compounds, some of the smaller molecular weight impurities found in lanes 5,7 and 9 are not present in the desalted fractions. 160 Chapter 7: Purification Protocol for BioLogic LP System
Protein Expression and Purification Series Purification Workflow for BioLogic LP System Affinity Purification of GST-DHFR-His Protein Follow the preceding protocols for your instrument and either handpacked columns or prepacked cartridge purificaton. 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 mircrocentrifuge 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. Chapter 7: Purification Protocol for BioLogic LP System BioLogic LP System Post-Purification: Choosing your flowthrough, wash, and three eluted GST-DHFR-His fractions 161 Flowthrough PAGE 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 + 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 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 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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Protein Expression and Purification Series - Bio-Rad

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