Protein Expression and Purification Series - Bio-Rad

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APPENDIX A TIPS AND HELPFUL HINTS Protein Expression and Purification Series 6. Affinity purification—Instrumentation protocols It is highly recommended that the starter kit (BioLogic LP Starter Kit catalog #731-8350EDU or BioLogic DuoFlow Starter Kit catalog #760-0135EDU) be run prior to performing the Protein Expression and Purification Series with your students. This will ensure the equipment is set up with the proper plumbing and electrical connections. 7. Size exclusion purification—Desalting columns A variable speed microcentrifuge is required for the desalting purification step. It is critical to set the variable speed microcentrifuge so that it generates 1,000 x g of force. • For the Bio-Rad Model 16K microcentrifuge, 1,000 x g is equivalent to a speed of 3,500 rpm. A Bio- Rad mini-centrifuge should NOT be used since these produce 2,000 x g of centrifugal force which is too much for the Bio-Gel® P–6 desalting resin to withstand. 8. DHFR enzymatic assay The DHFR enzymatic reaction occurs very quickly. Therefore, it is important to be familiar with the steps involved and to perform them quickly. A very minor change in the absorbance occurs at 340 nm with the conversion of NADPH to NADP+. This change is on the order of 0.05–0.08 OD in 150 seconds. Please note: • The DHF needs to be reconstituted in 10X PBS. • The NADPH needs to be reconstituted in 1X PBS. • The DHF and NADPH are NOT stable once reconstituted. They should be stored on ice and are only stable for three to four hours. The DHF and NADPH solutions cannot be frozen to retain or extend the activity and shelf life. The DHFR Enzymatic Assay Module can be purchased separately if more reactions are desired or if classes performing the assay are more than three to four hours apart. • Time is of utmost importance once the DHF is added to the 1x PBS, the GST-DHFR-His, and NADPH solution. Therefore, prior to the addition of the DHF to the cuvette containing the 1x PBS, the GST- DHRF-His sample and the NADPH, the spectrophotometer should be set up and ready to read. The DHF should be added while standing next to the instrument, mixed fast, placed in the instrument, and read immediately to ensure that an accurate measurement of the enzyme activity is captured. • If using trUView cuvettes please note that the frosted side contains the small clear window through which the UV reading is taken. 212 Appendix A: Tips and Helpful Hints
Protein Expression and Purification Series Appendix B: Results Analysis Centrifugation Purification Results Analysis The gel image below shows how typical results using the Centrifugation Purification Process should appear. Listed below are common steps that can help you analyze your results. 250 kD 150 100 75 50 37 25 20 15 10 1 2 3 4 5 6 7 8 9 Step 1: Check that induction occurred. The GST-DHFR-His protein runs on SDS-PAGE gels at an apparent MW of approximately 43 kD. (Note: the protein runs at this weight despite the actual protein molecular weight of 52 kD.) Results: An obvious band should appear in lane 3, the induced sample, between the 37 kD and 50 kD molecular weight marker if the GST-DHFR-His has been strongly induced. This band is not present in lane 2, the uninduced sample. Step 2: Check that lysis worked. Lanes 4 and 5 represent the insoluble and soluble fractions, respectively. Results: If the GST-DHFR-His was strongly induced and the cells lysed, a band of approximately 43 kD should be present in lane 5. It should be the same size as the band that occurred in lane 3 (falls between the 37kD and 50 kD molecular weight markers). Please note that the large band that is apparent at approximately 12 kD in lanes 4, 5, 6 and 7 is the lysozyme used to lyse the cells and not GST-DHFR-His. GST-DHFR-His is expressed in both an insoluble and soluble form. It is likely that there will be strong bands in both lanes 4 and 5 around 43 kD, representing both insoluble and soluble GST-DHFR-His respectively. As long as there is a strong band in lane 5, lysis has occurred, and soluble GST-DHFR-His has been released. Step 3: Check that the binding of the GST-DHFR-His occurred. Lane 6 is the flowthrough of proteins that did not bind to the Ni-IMAC resin. Results: There should be little to no 43 kD band visible in lane 6, the flowthrough, relative to lane 5, the soluble fraction, if the GST-DHFR-His bound well to the column. Step 4: Check that the GST-DHFR-His eluted from the column when elution buffer was added. Lane 8 is the GST-DHFR-His eluate fraction. Results: There should be a band that accounts for the majority of the protein in lane 8 of approximately 43 kD (falls between the 37 kD and 50 kD molecular weight markers). The purity and darkness of the GST-DHFR-His band will depend on the specific culture and induction times used, but the main peak seen in lane 8 should be the GST-DHFR-His. Appendix B: Purification Results Analysis 213 Figure 1. SDS-PAGE gel showing typical results from using the Centrifugation Purification Process. Lane 1, molecular weight markers; lane 2, uninduced sample; lane 3, induced sample; lane 4, insoluble fraction; lane 5, soluble fraction; lane 6, flowthrough; lane 7, wash; lane 8, GST-DHFR-His eluate fraction; lane 9, desalted GST-DHFR-His eluate fraction. GST-DHFR-His lysozyme APPENDIX B RESULTS ANALYSIS
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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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CHAPTER 6 HANDPACKING COLUMNs Prote
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CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
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Page 218 and 219: APPENDIX C RCF TO RPM CONVERSTION P
Page 220 and 221: APPENDIX D SETTING UP THE SMARTSPEC
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Page 224 and 225: APPENDIX E MINI-PROTEAN GEL BOX SET
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Page 230 and 231: APPENDIX G BIOMANUFACTURING Protein
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Page 234 and 235: APPENDIX H FOCUS QUESTIONS INSTRUCT
Page 242 and 243: APPENDIX I GLOSSARY Protein Express
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Page 246 and 247: APPENDIX J REFERENCES, LEGAL NOTES
Page 250: Life Science Group Bulletin 1665067
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Protein Expression and Purification Series - Bio-Rad

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