Protein Expression and Purification Series - Bio-Rad

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CHAPTER 4 11 ml CULTURE PROTOCOL Protein Expression and Purification Series Sample binding to the column 1. 2. 3. Attach a yellow tip closure to the bottom of the column. Add up to 600 µl of soluble fraction of the lysate to the column and attach the clear top cap onto the column. Gently mix the column for 20 minutes at room temperature on a tube rotator or a mini rocker. (A uniform slurry should be formed.) 4. Label a 2 ml microcentrifuge tube “ Flowthrough.” 5. 6. 7. Carefully twist and remove the yellow tip closure and place the column into the labeled 2 ml microcentrifuge tube. Remove the clear top cap from the column and centrifuge for 2 minutes at 1,000 x g to collect the flowthrough fraction. Cap and save the flowthrough fraction. Keep the column for the following steps. Washing the column 1. Label a 2 ml microcentrifuge tube “ Wash fraction.” 2. Place the column into the labeled 2 ml microcentrifuge tube. 3. Gently add 600 µl of wash buffer to the column and centrifuge for 2 minutes at 1,000 x g to collect the wash fraction. 4. Cap and save the wash fraction. Keep the column for the following steps. Elution of GST-DHFR-His protein from the column 1. Label a 2 ml microcentrifuge tube “ Eluate.” 2. Place the column into the labeled 2 ml microcentrifuge tube. 3. Gently add 400 µl of Elution buffer to the column and centrifuge for 2 minutes at 1,000 x g to collect the eluate fraction. 4. Cap and save the eluate fraction. Discard the column. Desalting of the GST-DHFR-His protein (eluate fraction) to remove imidazole 1. Invert the desalting column (with a green cap) sharply several times to resuspend the settled gel and to remove any bubbles. The resin should settle into the column and little to no resin should remain in the green cap. 2. 3. Snap off the bottom tab of the column and place the column into a clean 2 ml microcentrifuge tube. Remove the green top cap. If the column does not begin to flow, push the cap back on the column and then remove it again to start the flow. 102 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series 4. Allow the excess packing buffer to drain by gravity to the top of the gel bed (about 2 minutes), then place the column into a clean 2 ml tube. 5. Centrifuge for 2 minutes in a microcentrifuge at 1,000 x g (see Appendix C for more information about setting centrifuge speed) to remove the remaining packing buffer. Discard the buffer and the microcentrifuge tube. Keep the column for the following steps. 6. Label a clean 2 ml microcentrifuge tube “ Desalted eluate” with your initials and place the column into the 2 ml microcentrifuge tube. Carefully apply 75 µl of “Eluate” fraction directly to the center of the column. Be careful not to touch the resin with the pipet tip. 7. After loading the sample, centrifuge the column for 4 minutes at 1,000 x g. 8. Carefully apply another 75 µl of “Eluate” fraction directly to the center of the column, again being careful not to touch the resin with the pipet tip. 9. After loading the sample, centrifuge the column for 4 minutes at 1,000 x g. 10. You should now have approximately 150 µl of desalted eluate in the labeled tube. 11. Discard the column. Prepare SDS-PAGE samples 1. In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of flowthrough and label the tube “Flowthrough PAGE” with your initials. 2. 3. 4. 5. In a clean 1.5 ml screwcap microcentrifuge tube, combine 50 µl of Laemmli sample buffer with 50 µl of wash fraction and label the tube “Wash PAGE” with your initials. In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of Eluate and label the tube “Eluate PAGE” with your initials. In a clean 1.5 ml screwcap microcentrifuge tube, combine 25 µl of Laemmli sample buffer with 25 µl of desalted eluate and label the tube “Desalted Eluate PAGE” with your initials. Heat the Soluble PAGE, Flowthrough PAGE, Wash PAGE, Eluate PAGE and Desalted Eluate PAGE samples at 95°C for 5 minutes. 6. All SDS-PAGE samples can be stored at –20°C until SDS-PAGE analysis is to be performed. Storage of chromatography fractions The flowthrough, wash, eluate and desalted eluate fractions should all be stored at 4°C until the DHFR Enzymatic Assay is to be performed. Do not freeze the samples. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 103 CHAPTER 4 11 ml CULTURE 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 7 BIOLOGIC LP SYSTEM PROTOC
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CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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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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Protein Expression and Purification Series - Bio-Rad

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