Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Load, Run, Stain and Destain the Gel 1. Prepare samples: If samples have been stored at –20°C, reheat SDS-PAGE samples at 95°C for two minutes to redissolve any precipitated detergent and then centrifuge the samples for two minutes at 16,000 x g. 2. Assemble gel boxes: If using Bio-Rad Mini-PROTEAN Tetra gel boxes and TGX precast gels, see appendix E for more information on how to prepare gels and assemble gel boxes. 3. Load the gel: Using a fresh tip each time, load the following volumes of each sample into the appropriate well of your gel: 4. 5. 6. 7. 8. 9. Well Volume Sample 1 10 µl Precision Plus Protein Dual Color standard 2 10 µl Soluble PAGE 3 10 µl Flowthrough PAGE 4 20 µl Wash PAGE 5 20 µl Fraction A PAGE 6 20 µl Desalted A PAGE 7 20 µl Fraction B PAGE 8 20 µl Desalted B PAGE 9 20 µl Fraction C PAGE 10 20 µl Desalted C PAGE Run the gel at 200 V for 30 minutes. (If using a Bio-Rad Mini-PROTEAN TGX precast gel, the gel can be run at 300 V for 15 minutes.) After the run is complete, remove the gel from the cassette and place in the gel staining tray. Wash the gel three times with 50 ml of distilled water for five minutes each rinse. Discard all the rinse water. Note: Make sure that all of the wash water has been removed because excess water diluting the gel stain will interfere with staining efficiency. Stain the gel with 50 ml of Bio-Safe Coomassie stain for one hour. After one hour discard the Bio-Safe Coomassie stain and add 100 ml of distilled water and destain the gel overnight. Image the gel if you have an imaging system or dry the gel if you have a cellophane drying system. 10. Pick the desalted GST-DHFR-His fraction that has the highest concentration (darkest band) but also looks the most pure on the SDS-PAGE gel to analyze enzymatic activity (Figure 8.30). The most pure sample would show an obvious dark band for GST-DHFR-His and few or faint bands at other molecular weights in the lane on the SDS-PAGE gel. 194 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series 250 150 100 75 50 37 25 20 15 10 Figure 8.30. 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 which 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. Chapter 8: Purification Protocol for BioLogic DuoFlow System 1 2 3 4 5 6 7 8 9 10 Flowthrough Wash 195 Elute Column wash 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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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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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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