Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

APPENDIX H FOCUS QUESTIONS INSTRUCTORS GUIDE Protein Expression and Purification Series Focus Questions: Protein Purification Introduction 1. What is protein purification? Protein purification is the isolation of a specific protein from other molecules. The level of purity of the isolated protein is dictated by the subsequent use of the protein. 2. Describe lysis and why it is a necessary part of the protein purification process. Name a lysis technique. Lysis is the breaking open of the cell to release the components in the cytosol. For proteins that are not secreted into the culture medium, cells must be broken open to access the recombinant protein in the cytosolic space in order to begin to isolate that protein. Lysis techniques include freeze-thaw cycles, enzymatic lysis using lysozyme, and physical methods such as sonication and grinding. 3. 4. 5. Why is it important to remove DNA from the sample? It is important to remove genomic DNA from the sample because genomic DNA from lysed cells is highly viscous. If high-viscosity genomic DNA is flowed over a chromatography column during protein purification steps, it may clog the frit (bed support) that holds the chromatography resin in the column. What enzyme is used to break down the DNA in a lysate but leaves the protein intact? DNase. What is a common method of capture, or isolation, of protein? Chromatography. 236 Appendix H: Focus Question Answers
Protein Expression and Purification Series Focus Questions: Introduction to Chromatography 1. 2. 3. What is the purpose of chromatography? Chromatography separates biological molecules from complex mixtures using a specific property of the desired molecule such as its charge, hydrophobicity, size, or specific ability to bind or interact with other molecules. What is the mobile phase in chromatography? In chromatography, the mobile phase is the buffer or solvent containing the molecules to be separated. What is the stationary phase in chromatography? Give three examples of stationary phase. Stationary phase refers to the medium through which the mobile phase travels. Paper, glass beads, and resin beads are all examples of stationary phase. 4. Name four types of liquid chromatography techniques. 5. 6. Hydrophobic Interaction (HIC), size exclusion (SEC), ion exchange (IEX), mixed mode, and affinity (AC). What is an anion and what is a cation? When would you use anion exchange chromatography versus cation exchange chromatography? An anion is a negatively charged ion or molecule. Anion exchange chromatography utilizes a positively charged resin that can be used to bind negatively charged molecules. If the molecule of interest to be captured and purified is negatively charged, then anion exchange chromatography is used. A cation is a positively charged ion or molecule. Cation exchange chromatography utilizes a negatively charged resin that can be used to bind positively charged molecules. If the molecule of interest to be captured and purified is positively charged, then cation exchange chromatography is used. What is an affinity chromatography tag? Name two tags. An affinity chromatography tag is an amino acid sequence added to the recombinant protein that allows for that protein tag to be recognized and bound by an affinity chromatography method. Two types of tags are polyhistidine-tag and GST-tag. 7. Does adding an affinity tag to a protein change the protein? Explain your answer. Yes, it does. It is the addition of amino acids and this may or may not interfere with the protein's ability to fold correctly and/or function properly. Appendix H: Focus Question Answers 237 APPENDIX H FOCUS QUESTIONS INSTRUCTORS GUIDE
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:
Page 84 and 85:
Page 86 and 87:
Page 88 and 89:
Page 90 and 91:
Page 92 and 93:
CHAPTER 4 11 ml CULTURE PROTOCOL Pr
Page 94 and 95:
Page 96 and 97:
Page 98 and 99:
Page 100 and 101:
Page 102 and 103:
Page 104 and 105:
Page 106 and 107:
Page 108 and 109:
Page 110 and 111:
Page 112 and 113:
Page 114 and 115:
Page 116 and 117:
Page 118 and 119:
CHAPTER 5 100 ml CULTURE PROTOCOL P
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Page 134 and 135:
CHAPTER 6 HANDPACKING COLUMNs Prote
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 142 and 143:
Page 144 and 145:
Page 146 and 147:
Page 148 and 149:
Page 150 and 151:
Page 152 and 153:
Page 154 and 155:
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: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 202 and 203: CHAPTER 9 DHFR ENZYMATIC ASSAY Prot
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 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

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?