Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

APPENDIX H FOCUS QUESTIONS INSTRUCTORS GUIDE Protein Expression and Purification Series Focus Questions: Centrifugaton and Chromatography Instrumentation Purification 1. 2. What is gravity chromatography? Name an advantage and disadvantage of this system. Gravity chromatography uses gravity (via nature or centrifugation) to pull the liquid phase through the solid phase. Advantages of gravity chromatography are that it’s inexpensive and does not require any special laboratory equipment. Disadvantages of gravity chromatography are time and the requirement that someone “baby-sit” the experiment. Is centrifugation/spin chromatography the same as gravity chromatography? Explain your answer. Centrifugation/spin chromatography and gravity chromatography both use gravitational force to push fluids through chromatography columns. Gravity chromatography is limited to 1 x g of force. Centrifugation/spin chromatography controls the g-force by the speed of the centrifuge. Higher speeds exert more g-force and can increase the flow rate. One difference between the two methods is that centrifugation/spin chromatography requires a centrifuge and columns that fit into that centrifuge. As a result, gravity chromatography columns can have a larger variety of lengths and diameters than centrifugation/spin chromatography columns. 3. What are the advantages of spin chromatography? When would you choose to use centrifugation/spin chromatography and why? 4. 5. Spin chromatography is quick, and the spin columns fit in a standard bench-top centrifuge and are available both empty and prepacked. If the size of column volume and resin can be accommodated, a centrifuge can generate several thousand g-force. This increased force applied to the mobile phase will pull it through the resin more quickly than 1 x g of force for a gravity column. If the resin packed in a chromatography column can withstand higher forces without being crushed, a spin column can be faster than a gravity column. What are the advantages of using prepacked cartridges? When would you choose to use prepacked cartridges and why? Prepacked cartridges are manufactured to have evenly packed resin beds with no air pockets or channels. There is a lot of uniformity of performance between cartridges. Large scale chromatography columns used in the production of biopharmaceuticals are always handpacked due to the column size and amount of resin used. Prepacked cartidges are typically only used for bench-scale purifications. What is a fraction in chromatography? A fraction is a sample of known volume of eluate from a chromatography column. 238 Appendix H: Focus Question Answers
Protein Expression and Purification Series Focus Questions: SDS-PAGE Electrophoresis 1. What are four ways to quantify the amount of protein in a sample? 1, Absorbance at 280 nm using a spectrophotometer; 2, Bradford or Lowry protein assay; 3, ELISA; 4, SDS-PAGE or polyacrylamide gel analysis. 2. What does primary (1°), secondary (2°), tertiary (3°), and quaternary (4°) protein structure refer to? Primary protein structure refers to the amino acid sequence of the protein. Secondary protein structure refers to the hydrogen bonds that link the amino acids and cause pleating or spiraling. Tertiary structure refers to the 3-D structure of the protein as it is folded up. Quaternary structure refers to the protein complex—more than one amino acid chain, or subunit, joined to make a functional protein. 3. Describe SDS-PAGE analysis. Why would you use SDS-PAGE to analyze your samples? Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate a mixture of proteins based solely on protein size with the use of a gel medium and an electric field. The relative quantities of different proteins as well as the sizes of those proteins can be visualized using SDS-PAGE. Therefore, for protein expression and purification, because the size of the recombinant protein produced is known, SDS-PAGE can be used to visualize if the protein is expressed, soluble or insoluble, and how well it was purified. 4. What are BME and DTT and what do they do to a protein? BME, β-mercaptoethanol, and DTT, dithiothreitol, are reducing agents that disrupt a protein’s disulfide bonds that help stabilize the structure of some proteins. 5. Describe the components and function of each component of Laemmli sample buffer. Laemmli sample buffer is a commonly used SDS-PAGE sample buffer. It consists of Tris to maintain pH conducive to electrophoresis, glycerol to weigh the sample, SDS to equalize protein charge, potentially a reducing agent to break protein disulfide bonds, and bromophenol blue, which gives the sample color. Appendix H: Focus Question Answers 239 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:
Page 188 and 189: 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

Create successful ePaper yourself

Delete template?

Save as template?