Protein Expression and Purification Series - Bio-Rad

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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOCOL Protein Expression and Purification Series Quantitation of Protein in Desalted Fractions Student Workstations Each student team requires the following items to quantitate the amount of purified, desalted eluate (GST-DHFR-His) it has via spectroscopy: Material Needed for Each Workstation Quantity Desalted fractions (150 µl each) 3 20–200 µl adjustable-volume micropipet and tips 1 trUView disposable cuvettes (or UV compatible cuvettes) 3 Marking pen 1 Common Workstation Quantity UV spectrophotometer 1–2 Protocol: Quantitation of Protein in Desalted Fractions 1. 2. Make sure that your cuvettes are completely clean and dry before use. 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 fractions A, B, and C each into its own clean cuvette. 5. 6. Measure the absorbance at 280 nm of all your desalted fractions. A 280 Desalted fraction A __________________________ A 280 Desalted fraction B __________________________ A 280 Desalted fraction C __________________________ Pipet each desalted fraction back into the tube containing that fraction after measuring its absorbance. 7. Make sure that you have at least 15 µl of desalted GST-DHFR-His to run your enzyme assay. 8. Calculate the concentration of GST-DHFR-His in your desalted fractions 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 156 Chapter 7: Purification Protocol for BioLogic LP System
Protein Expression and Purification Series The concentration of GST-DHFR-His (M) = Absorbance/75,540 Concentration of GST-DHFR-His (Desalted fraction A) = ______________________ M Concentration of GST-DHFR-His (Desalted fraction B) = ______________________ M Concentration of GST-DHFR-His (Desalted fraction C) = ______________________ M B: Convert from molarity to mg/ml for the amount of GST-DHFR-His in your purified desalted fractions 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 (Desalted fraction A) = _________________________mg/ml Concentration of GST-DHFR-His (Desalted fraction B) = _________________________mg/ml Concentration of GST-DHFR-His (Desalted fraction C) = _________________________mg/ml These are the concentrations of GST-DHFR-His that you produced and purified and are contained in your 150 µl desalted eluate fractions. Chapter 7: Purification Protocol for BioLogic LP System 157 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 9 DHFR ENZYMATIC ASSAY Prot
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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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