Protein Expression and Purification Series - Bio-Rad

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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL Protein Expression and Purification Series Section 2. Affinity Chromatography using Native IMAC Protocol to Purify GST- DHFR-His Manually preparing the DuoFlow system and IMAC Ni-charged column 1. Prime the Pump Inlet tubing with Buffer A and Buffer B The inlet tubing is currently filled with water. At this point, the inlet tubing will be primed with Buffer A and Buffer B in preparation for cleaning and equilibrating the column. 1. 2. 3. 4. 5. Ensure the pumps are stopped and the AVR7-3 inject valve is in the Load (L) position on the Manual screen. Place the tubing for Pump A into the container of Buffer A. Make sure that the tubing sits near the bottom of the container. Tape the tubing down if necessary. Place the tubing for Pump B into the container of Buffer B. Make sure that the tubing sits near the bottom of the container. Tape the tubing down if necessary. Attach the 10 ml syringe to the priming port for Pump A, turn the priming port counterclockwise to open, and pull 1–2 ml of Buffer A into the syringe to remove any air bubbles introduced into the Inlet line. Turn the priming port clockwise to close and remove the syringe. Expel the buffer in the syringe into the waste beaker. Repeat step 4 for priming port B. 2. Cleaning and equilibrating the IMAC Ni-charged column in Manual mode At this point, the lines throughout the entire system will first be filled with Buffer B (20 mM sodium phosphate buffer, 300 mM NaCl, 250 mM imidazole, pH 8) to wash any remaining contaminants off of the IMAC column. Then the lines will be filled with 2% Buffer B (20 mM sodium phosphate buffer, 300 mM NaCl, 5 mM imidazole) to flush the 100% Buffer B solution out of the system and replace it with 2% Buffer B (the same composition buffer that the soluble fraction is in). The column will be equilibrated at this point and ready for use. 1. 2. Set the AVR7-3 inject valve to position L (Load). Set the Gradient Pump Flowrate to 2.00 ml/min, Inlet B to 100%, High limit to 200 psi and Low limit to 0 psi, then press the Start button (see Figure 8.12). Allow the wash to proceed for five minutes. While the pump is running, continue to steps 4–6. Figure 8.12. Manual wash. The column and tubing are first washed with 100% Buffer B at 2.00 ml/min for five minutes. In chromatography nomenclature, this is a 10 CV (column volume) wash since 10 ml of buffer are flowed over a 1 ml column. This step washed off any storage buffer remaining on the column and any residue from previous purifications (if present). 174 Chapter 8: Purification Protocol for BioLogic DuoFlow System
Protein Expression and Purification Series 3. While the pump is running, look at the GP-Pressure curve (Figure 8.13) and determine if the pressure is even. 4. While there might be small oscillations, there should be no large variances. If there are large fluctuations (such as going from 50 psi to 18 psi and back), this means there are air bubbles in the lines and you will need to stop. Stop the pump. Prime Inlet line B again, following the protocols in Step 1 and then start Pump B again. 5. Once there are no air bubbles in the line (no pressure fluctuations), record the UV absorbance and conductivity values from the bottom of the Manual screen and then stop the pump. UV _______________________ Conductivity _____________________________ Figure 8.13. Manual wash with 100% B. Imidazole absorbs at 280 nm so you should see a UV value less than 0.5. Buffer B contains a high level of salt (300 mM NaCl and 20 mM sodium phosphate) that contributes to the main portion of the conductivity value. Imidazole contributes some conductivity, but is not a strong ionic species and hence contributes much less to the overall conductivity. The conductivity range expected after the 100% Buffer B wash is between 35–55 mS/cm. The chromatogram shows equilibration of the UV trace, conductivity trace, and pressure reading with the curves that go flat (note the Y-axes have been expanded for easier viewing). 6. Set the buffer composition to 98% Inlet A and 2% Inlet B. Leave the flow rate at 2.00 ml/min and the high and low pressure limits at 200 psi and 0 psi respectively. 7. Press the Start button and equilibrate the system for five minutes (or 10 CV). 8. While the pump is running, look at the GP-Pressure curve (Figure 8.14) and determine if the pressure is even. 9. While there might be small oscillations, there should be no large ones. If there are large fluctuations (such as going from 50 psi to 18 psi and back), this means there are air bubbles in the lines and you will need to stop the pumps. Prime Inlet line A again following the protocols in Step 1 and then start the pumps again. 10. After five minutes of equilibration with 2% Buffer B, record the UV and Conductivity values and then stop the pump. Chapter 8: Purification Protocol for BioLogic DuoFlow System 175 % B Conductivity UV Pressure UV _______________________ Conductivity _____________________________ 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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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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