Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

CHAPTER 5 100 ml CULTURE PROTOCOL Protein Expression and Purification Series Chromatography Instrumentation Purification Workflow Separating Soluble from Insoluble Induced Cell Fractions and Preparation of SDS-PAGE Samples Separate soluble from insoluble cell lysate components. Transfer thawed, lysed cells to a 30–50 ml centrifuge tube. Balance centrifuge and centrifuge for 20 minutes at 16,000 x g. Label 50 ml sterile conical tube Soluble fraction with your initials. The supernatant contains the soluble fraction, and the pellet contains the insoluble fraction. Gently pour the supernatant into tube labeled Soluble fraction. Relabel the Lysed Cells tube Insoluble fraction . Soluble fraction Prep Insoluble PAGE sample Spin 20 min at 16,000 x g Add 5 ml Equilibration buffer and 5 ml 2x PBS to the Insouble fraction. + 5 ml + 5 ml Equilibration 2x PBS buffer Insoluble fraction The insoluble pellet contains a large quantity of genomic DNA, making it viscous and difficult to resuspend. Using a syringe with 22 gauge needle, repeatedly pull the solution through the syringe to shear the pellet and decrease the viscosity. Label two clean 1.5 ml screwcap microcentrifuge tubes Soluble PAGE and Insoluble PAGE. Take a 50 µl aliquot from each fraction Soluble fraction and Insoluble fraction and prepare for SDS-PAGE per instruction manual. Store the SDS-PAGE samples, Soluble and Insoluble fractions at –20°C until ready for further steps. Insoluble Fraction 50 µl Lysed cells Decant supernatant into tube labeled Soluble fraction Shear insoluble fraction with syringe needle to resuspend pellet Soluble Fraction 50 µl Prep Soluble PAGE sample 132 Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification
Protein Expression and Purification Series Chromatography Instrumentation Purification Workflow SDS-PAGE Electrophoresis Analysis of Induction Load, run, stain and destain the gel. Reheat SDS-PAGE samples at 95°C for two minutes. Centrifuge samples for two minutes at 16,000 x g. Using a fresh tip each time, load the following volumes of each sample into the appropriate well of your gel Lane Volume Sample 1 10 µl Precision Plus Dual Color standard 2 7.5 µl Uninduced PAGE 3 15 µl Induced PAGE 4 10 µl Insoluble PAGE 5 10 µl Soluble PAGE 6 - 10 Empty (or a second lab group can use these wells to load their samples) Run the gel at 200 V for 30 minutes. (If using 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. Stain the gel with 50 ml of Bio-Safe Coomassie stain for one hour. After one hour, discard the Bio-Safe Coomassie stain, 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. Chapter 5: 100 ml Culture Protocol for Chromatography Instrumentation Purification 133 Heat 2 min at 95°C Spin 2 min at 16,000 x g CHAPTER 5 100 ml CULTURE PROTOCOL
Page 1:
Biotechnology Explorer TM Protein E
Page 4 and 5:
Protein Expression and Purification
Page 6 and 7:
Page 8 and 9:
Page 10 and 11:
Page 12 and 13:
Page 14 and 15:
Page 16 and 17:
Page 18 and 19:
Page 20 and 21:
Page 22 and 23:
Page 24 and 25:
Page 26 and 27:
Page 28 and 29:
Page 30 and 31:
Page 32 and 33:
Page 34 and 35:
CHAPTER 1 BACKGROUND Protein Expres
Page 36 and 37:
Page 38 and 39:
Page 40 and 41:
Page 42 and 43:
Page 44 and 45:
Page 46 and 47:
Page 48 and 49:
Page 50 and 51:
CHAPTER 2 FOCUS QUESTIONS Protein E
Page 52 and 53:
Page 54 and 55:
Page 56 and 57:
CHAPTER 3A ADVANCE PREP CENTRIFUGAT
Page 58 and 59:
Page 60 and 61:
Page 62 and 63:
Page 64 and 65:
Page 66 and 67:
Page 68 and 69:
Page 70 and 71:
Page 72 and 73:
CHAPTER 3B ADVANCE PREP INSTRUMENTA
Page 74 and 75:
Page 76 and 77:
Page 78 and 79:
Page 80 and 81:
Page 82 and 83: CHAPTER 3B ADVANCE PREP INSTRUMENTA
Page 92 and 93: CHAPTER 4 11 ml CULTURE PROTOCOL Pr
Page 118 and 119: CHAPTER 5 100 ml CULTURE PROTOCOL P
Page 134 and 135: CHAPTER 6 HANDPACKING COLUMNs Prote
Page 140 and 141: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 182 and 183:
CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 184 and 185:
Page 186 and 187:
Page 188 and 189:
Page 190 and 191:
Page 192 and 193:
Page 194 and 195:
Page 196 and 197:
Page 198 and 199:
Page 200 and 201:
Page 202 and 203:
CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
Page 204 and 205:
Page 206 and 207:
Page 208 and 209:
Page 210 and 211:
APPENDIX A TIPS AND HELPFUL HINTS P
Page 212 and 213:
APPENDIX A TIPS AND HELPFUL HINTS P
Page 214 and 215:
APPENDIX B RESULTS ANALYSIS Protein
Page 216 and 217:
APPENDIX B RESULTS ANALYSIS Protein
Page 218 and 219:
APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221:
APPENDIX D SETTING UP THE SMARTSPEC
Page 222 and 223:
APPENDIX D SETTING UP THE SMARTSPEC
Page 224 and 225:
APPENDIX E MINI-PROTEAN GEL BOX SET
Page 226 and 227:
APPENDIX F BIOLOGIC LP SYSTEM SETUP
Page 228 and 229:
APPENDIX F BIOLOGIC LP SYSTEM SETUP
Page 230 and 231:
APPENDIX G BIOMANUFACTURING Protein
Page 232 and 233:
APPENDIX G BIOMANUFACTURING Protein
Page 234 and 235:
APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 236 and 237:
Page 238 and 239:
Page 240 and 241:
Page 242 and 243:
APPENDIX I GLOSSARY Protein Express
Page 244 and 245:
APPENDIX I GLOSSARY Protein Express
Page 246 and 247:
APPENDIX J REFERENCES, LEGAL NOTES
Page 250:
Life Science Group Bulletin 1665067
show all

Protein Expression and Purification Series - Bio-Rad

Create successful ePaper yourself

Delete template?

Save as template?