Protein Expression and Purification Series - Bio-Rad

More documents

Recommendations

Info

CHAPTER 6 HANDPACKING COLUMNs Protein Expression and Purification Series CHAPTER 6: Handpacking a Glass Econo-Column Column Attached to a Flow Adaptor Student Workstations If students are handpacking their own columns they will need to use the following protocol. Otherwise, if they are using prepacked cartridges, this section can be skipped. Material Needed for Each Workstation Quantity Glass Econo-Column column 1 cm x 5 cm 1 Profinity IMAC Ni-charged resin slurry (50% slurry) 2 ml slurry Flow adaptor for 1 cm ID columns* 1 Two-way stopcock 1 10 ml syringe 1 Beaker with distilled water 1 Beaker for waste 1 100–1,000 µl adjustable-volume micropipet and tips 1 Marking pen 1 *Note: For complete instructions on using the flow adaptor, please see the instruction manual included with the product. Introduction The Econo-Column column flow adaptor provides a number of advantages in column chromatography. Specifically, for purification of GST-DHFR-His it can be used for: • Sample application with minimal dilution • More efficient elution of GST-DHFR-His, because the 250 mM imidazole stock solution is not diluted when applied to the column The Econo-Column column flow adaptors are shipped preassembled and ready to use. Figure 6.1 shows the parts of the assembled flow adaptor. Figure 6.1. Econo-Column column flow adaptor. 134 Chapter 6: Handpacking a Glass Econo-Column Column Attached to a Flow Adaptor
Protein Expression and Purification Series Flow Adaptor Description The cam latch can be adjusted to three positions allowing the flow adaptor to be situated into a glass column, and the O-ring to be tightened to seal the flow adaptor securely into the column so that there is no movement once the flow adaptor has been set up (Figure 6.2). Fitting the Flow Adaptor to the Glass Econo-Column column Due to normal tolerances in glass diameters, each flow adaptor should be adjusted to fit to individual columns. This will ensure optimal performance. 1. Pull the white flow adaptor body all the way up into the collar of the black flow adaptor body (See Figure 6.3). Figure 6.3. Preparing the flow adaptor to be fit into a glass Econo-Column column. 2. Attach the flow adaptor body to the column by sliding the flow adaptor body onto the yellow flange on the top of the glass column (Figure 6.4). Figure 6.4. Attaching the flow adaptor to the glass Econo-Column column. A) Look for the groove on the end of the black flow adaptor body. B) Line up the groove on the black flow adaptor body with the top of the yellow flange on the column. C) Gently slide the flow adaptor onto the glass column. D) The flow adaptor should snap into place when completely positioned on the top of the column. Chapter 6: Handpacking a Glass Econo-Column Column Attached to a Flow Adaptor Figure 6.2. The three positions of the cam latch and the corresponding effects on the O-ring. When the cam latch is in position 1, there is little pressure on the O-ring (A), and it should slide with ease through the glass column. When the cam latch is in position 2, the O-ring seals against the glass column wall (B) with a small amount of movement possible for final height adjustments. When the cam latch is in position 3, the O-ring completely seals the flow adaptor into position (C). A B C 135 CHAPTER 6 HANDPACKING COLUMNS
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: 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 136 and 137: CHAPTER 6 HANDPACKING COLUMNs Prote
Page 138 and 139: CHAPTER 6 HANDPACKING COLUMNs Prote
Page 140 and 141: CHAPTER 7 BIOLOGIC LP SYSTEM PROTOC
Page 166 and 167: CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
Page 184 and 185:
CHAPTER 8 BIOLOGIC DUOFLOW PROTOCOL
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

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

Delete template?

Save as template?