Protein Expression and Purification Series - Bio-Rad

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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOCOL 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 LP DataView software on the chromatogram where the peak eluted are the specific fraction tubes where the protein can be found. However, 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 Figures 7.14 and 7.15, according to the chromatogram, the protein should have eluted in fractions 10–11. However, 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 7.14 Example chromatogram of a run. Figure 7.15. Magnified region where the GST-DHFR-His eluted. 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 152 Chapter 7: Purification Protocol for BioLogic LP System
Protein Expression and Purification Series ahead of time by attaching a syringe to the fitting at the inlet of the UV detector when the tubing is filled with fluid and pushing all this fluid out through the fraction collector drop former and measuring the volume. Using the calculation method, a loose estimate can be determined by calculating the delay volume of the tubing present. The silicone tubing has an inner diameter (ID) of 0.16 cm. The volume of fluid in a specific length (L) of tubing would be π x (ID/2) 2 x L. Therefore, as an example, assuming that 1.6 mm ID tubing is in place and that there are 30 cm of tubing between the UV detector and the fraction collector, the delay volume would be at a minimum: π x (0.16 cm/2) 2 x 30cm = 0.6 cm 3 or 0.6 ml. As can be seen from the calculation, this is a volume equivalent to about half of the eluted fraction volume size. However, there are also delay volumes as part of the UV flow cell, conductivity flow cell and fittings. Since 1 ml fractions are being collected for the eluate, it is important not to discard any fractions until all analyses are done! In the case of the example shown above, the majority of the GST-DHFR-His has eluted in fractions 11, 12 and 13. The highest concentration was in fraction 12, which would represent a delay volume of approximately 2 ml. Two other fractions will also be examined. 1) The flowthrough fraction that contains all proteins that did not bind to the column when the column was initially loaded with the soluble fraction, and 2) the wash fraction that contains any proteins that were washed off when the imidizole level was increased to 4% (or 10 mM imidazole). Again, these fractions can be determined by looking at the chromatogram from the BioLogic LP system run. For example, in Figure 7.14, if delay volume is considered, the majority of the flowthrough fraction should be in fraction 2. The majority of the wash fractions would be in fractions 5-8 in the example chromatogram in Figures 7.14 and 7.15. Protocol Choosing your flowthrough, wash, and three eluted GST-DHFR-His fractions Examine your chromatogram and determine which fractions are most likely to contain your flowthrough and wash fractions, and three fractions which might contain your eluted GST-DHFR-His. Remember that the delay volume is probably between 1–3 ml depending on the tubing length of your instrument. Record the five fraction numbers below. Flowthrough fraction : ____________________ Wash fraction: __________________________ First eluted GST-DHFR-His fraction: ________________________________ Second eluted GST-DHFR-His fraction: ______________________________ Third eluted GST-DHFR-His fraction: _______________________________ Chapter 7: Purification Protocol for BioLogic LP System 153 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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CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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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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