Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

CHAPTER 4 11 ml CULTURE PROTOCOL Protein Expression and Purification Series Quantitation of Protein in Desalted Fraction Student Workstations Each student team requires the following items to quantitate the amount of purified, desalted eluate (GST-DHFR-His) they have via spectroscopy: Material Needed for Each Workstation Quantity Desalted eluate sample 150 µl 20–200 µl adjustable-volume micropipet and tips 1 trUView disposable cuvette (or similar UV compatible cuvettes) 1 Marking pen 1 Common Workstation Quantity UV spectrophotometer 1–2 Protocol: Quantitation of Protein in Desalted Fraction 1. Make sure that your cuvettes are completely clean and dry before usage. 2. Set your spectrophotometer to read at 280 nm Note: If using a Bio-Rad SmartSpec Plus spectrophotometer, please see Appendix D for setup instructions. 3. Blank your spectrophotometer with 100 µl distilled water. 4. Pipet 100 µl of your desalted eluate sample into a clean cuvette. 5. Measure the absorbance at 280 nm of your desalted eluate sample. A 280 Desalted eluate sample: __________________________ 6. Pipet the sample from the cuvette back into your tube of desalted eluate sample. Make sure that you have at least 15 µl of desalted eluate sample to run your enzyme assay. 7. Calculate the concentration of GST-DHFR-His in your desalted eluate fraction as follows: A. The extinction coefficient (ε) of the entire GST-DHFR-His construct is theoretically calculated to be 75,540 M -1 cm -1 . Knowing that Absorbance = ε x C x L, where ε is 75,540 M -1 cm -1 L is the pathlength of the cuvette in cm (usually 1) and the absorbance at 280 nm is being measured The concentration of GST-DHFR-His (M) = Absorbance/75,540 Concentration of GST-DHFR-His = ______________________ M 104 Chapter 4: 11 ml Culture Protocol for Centrifugation Purification
Protein Expression and Purification Series B. Convert from molarity to mg/ml for the amount of GST-DHFR-His in your purified desalted eluate fraction knowing that the molecular mass of GST-DHFR-His is 50,361 g/mol. Concentration GST-DHFR-His (mg/ml) = Concentration GST-DHFR-His (M) x 50,361 g/mol Concentration of GST-DHFR-His = _________________________mg/ml This is the concentration of GST-DHFR-His that you produced and purified and is contained in your 150 µl desalted eluate fraction. Chapter 4: 11 ml Culture Protocol for Centrifugation Purification 105 CHAPTER 4 11 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:
CHAPTER 2 FOCUS QUESTIONS Protein E
Page 54 and 55: CHAPTER 2 FOCUS QUESTIONS Protein E
Page 56 and 57: CHAPTER 3A ADVANCE PREP CENTRIFUGAT
Page 72 and 73: 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 154 and 155:
CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 156 and 157:
Page 158 and 159:
Page 160 and 161:
Page 162 and 163:
Page 164 and 165:
Page 166 and 167:
CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 168 and 169:
Page 170 and 171:
Page 172 and 173:
Page 174 and 175:
Page 176 and 177:
Page 178 and 179:
Page 180 and 181:
Page 182 and 183:
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?