Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series 6. 7. 8. 9. In manual mode, set the buffer composition to a mix of 50% Buffer A and 50% Buffer B. Set the flow rate to 5 ml/min. Make sure that the waste beaker is empty and that the waste tubing flows into the waste beaker. In manual mode, press the Start button and allow the flow to continue for five minutes. 10. After five minutes, change the AVR7-3 valve to the Inject (I) position to allow flow through the sample loop. 11. 12. 13. Continue flow for another five minutes. Stop the pump. If requested by your instructor, repeat steps 3–11 using 20% ethanol in place of high-quality water. 14. Your instrument is now ready for storage or use by another student group. 186 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series Removing Imidazole (Desalting) from the Purified GST-DHFR- His and Preparing Samples for SDS-PAGE Analysis Student Workstations Each student team requires the following items to desalt three of their fractions in which they think their GST-DHFR-His samples reside and to prepare SDS-PAGE samples: Material Needed for Each Workstation Quantity Chromatogram from GST-DHFR-His purification 1 Fractions from GST-DHFR-His purification varies Desalting column 3 Screwcap microcentrifuge tubes, 1.5 ml 6 Laemmli buffer (left over from previous activity) 1 ml Microcentrifuge tubes, 2 ml 6–12 20–200 µl adjustable-volume micropipet and tips 1 Marking pen 1 Common Workstation Quantity Microcentrifuge with variable speed setting >16,000 x g 1 Dry bath or water bath set at 95°C 1 At this point, there might be several fractions that contain the eluted GST-DHFR-His protein. It is important to determine which fractions the purified GST-DHFR-His is present in and which fraction(s) have the highest concentration of GST-DHFR-His. It might be assumed that the fractions marked by the DuoFlow software on the chromatogram where the peak eluted are the specific fraction tubes where the protein can be found. However, it should be noted, that there is a time delay from when the absorbance of the fraction is measured on the UV detector to when it flows through the tubing past the conductivity meter and out into the fraction collector. This is called a delay time (or volume). For example, in Figure 8.29, according to the chromatogram, the protein should have eluted in fractions 7–8. Again, this would be assuming that there is no delay between when the protein is detected in the UV detector and when it drops into a fraction collector tube. Figure 8.29. Chromatogram of a run. If there was no delay volume between the UV detector and fraction collector, the GST-DHFR- His would be assumed to be in fractions 7, 8, and 9. However, there is a finite delay time between detection and collecting that can be calculated or measured. The delay time (volume) can be calculated by measuring the length of tubing present between the detector and the fraction collector as well as knowing the volume of all fittings in between the UV monitor and the fraction collector. It can also be measured experimentally ahead of time by attaching a syringe to the fitting Chapter 8: Purification Protocol for BioLogic DuoFlow System 187 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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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
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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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