Protein Protocols Protein Protocols

More documents

Recommendations

Info

712 Courchesne and Patterson Fig. 1. Flow chart outlining methods for obtaining peptides from gel-separated proteins by either digestion in-gel or on-membrane for subsequent MS analysis. In all cases, the spot (or band) of interest, together with a control piece containing no protein, is excised and subjected to digestion and extraction of the peptides. For the Immobilon-CD protocol, the washing and chopping into small pieces steps can be reversed. homologue) exists in the database. The caveats to this approach and considerations for interpretation of the results are described. Finally, we cover generation and analysis of sequence-specific fragment ions using post-source decay MALDI-MS. Either partially interpreted or uninterpreted fragment ion data can be used in search programs to identify proteins with high accuracy in sequence databases, and the use of this approach is also described. 2. Materials 2.1. Equipment 1. Water bath (that can be set at 25°C or 37°C). 2. Sonication bath. 3. Vacuum concentrator (e.g., Speed Vac, Savant).
MALDI-MS for <strong>Protein</strong> Identification 713 4. Polypropylene microcentrifuge tubes (500 µL). 5. MALDI-MS instrument (linear and/or reflector capability). 6. MALDI-MS instrument fitted with a reflector capable of variable voltage or a curvedfield reflector (the example employs the Kompact MALDI IV—a curved-field reflector MALDI-MS ([Kratos Analytical]). 2.2. General Reagents 1. 50% v/v methanol (MeOH) containing 0.1% v/v aqueous trifluoroacetic acid (TFA). 2. Deionized water (dH2O). 2.3. Enzymatic Digestion In-Gel 1. 50 mM Citric acid. 2. 20 mM NH4HCO3 containing 50% v/v acetonitrile (MeCN). 3. Stock enzyme: Endoproteinase LysC (5-µg vial, sequencing grade) (Boehringer Manheim) (LysC). 4. Digest buffer: 20 mM NH4HCO3, 1.0 mM CaCl2. 5. Laboratory rotating mixer. 6. 1% TFA. 7. 60% v/v MeCN containing 0.1% v/v aqueous TFA. 2.4. Enzymatic Digestion on Immobilon-CD 1. 20 mM Tris-HCl, pH 9.0, containing 50% v/v MeOH. 2. 25 mM Tris-HCl, 1 mM EDTA, pH 8.0. 3. Stock enzyme (LysC): As for Subheading 2.3., item 3. 4. 30% v/v MeCN containing 2.5% v/v aqueous TFA. 5. 60% v/v MeCN containing 2.5% v/v aqueous TFA. 2.5. Enzymatic Digestion on Immobilon-P 1. 70% v/v Aqueous MeCN. 2. Digest buffer: 25 mM NH4CO3, 1% octyl-β-glucoside, 10% v/v MeOH. 3. Stock enzyme (LysC): As for Subheading 2.3., item 3. 4. 50% v/v Ethanol–50% v/v formic acid (98% HCOOH) (prepared immediately prior to use). 5. 10% v/v Aqueous MeCN. 2.6. Microcolumn Chromatography 1. µ-Guard column, 300 µm internal diameter (i.d.) × 1 mm, C8 packing, 300 Å wide-pore (LC Packings) (see Note 1). 2. Lite-Touch ® ferrules for 1/16-in-outer diameter (o.d.) tubing. 3. Inlet tubing, 2 cm, 300 µm i.d., 1/16-in. o.d. Teflon ® tubing (PE-Applied Biosystems Division) or similar. 4. Outlet tubing, 2 cm, 0.005-in. i.d., 1/16-in. o.d. PEEK (Red)(Upchurch Scientific). 5. 10-µL Hamilton syringe, with a fixed or removeable beveled needle and Chaney adaptor (Baxter Healthcare). 6. 1% v/v Aqueous 98% HCOOH (make fresh weekly). 7. 10% v/v Increments, or as desired, of MeCN to 90% in 1% v/v Aqueous 98% HCOOH (make fresh weekly). 8. MeOH. 9. Parafilm.
Page 1 and 2:
The Protein Protocols Handbook SECO
Page 3 and 4:
The Protein Protocols Handbook SECO
Page 5 and 6:
Preface The Protein Protocols Handb
Page 7 and 8:
viii Contents 14 Identification of
Page 9 and 10:
x Contents 47 Conjugation of Fluoro
Page 11 and 12:
xii Contents 80 Peptide Mapping by
Page 13 and 14:
xiv Contents 112 Sialic Acid Analys
Page 15 and 16:
xvi Contents 145 Purification of Ig
Page 17 and 18:
Contributors THOMAS E. ADRIAN • D
Page 19 and 20:
Contributors xxi RUTH R. FRENCH •
Page 21 and 22:
Contributors xxiii STEFANIE A. NELS
Page 23 and 24:
UV Absorption 1 PART I QUANTITATION
Page 25 and 26:
4 Aitken and Learmonth 2. Materials
Page 27 and 28:
6 Aitken and Learmonth References 1
Page 29 and 30:
8 Waterborg 3. Method 1. To 0.1 mL
Page 31 and 32:
BCA for Protein Quantitation 11 3 T
Page 33 and 34:
BCA for Protein Quantitation 13 Tab
Page 35 and 36:
The Bradford Method 15 4 The Bradfo
Page 37 and 38:
The Bradford Method 17 Fig. 1. Vari
Page 39 and 40:
The Bradford Method 19 Table 2 Comp
Page 41 and 42:
The Bradford Method 21 13. Kirazov,
Page 43 and 44:
24 Akins and Tuan passes. The side
Page 45 and 46:
26 Akins and Tuan Fig. 2. Effect of
Page 47 and 48:
28 Akins and Tuan protein concentra
Page 49 and 50:
30 Akins and Tuan energy. Too much
Page 51 and 52:
32 Boerner et al. Several approache
Page 53 and 54:
34 Boerner et al. Fig. 2. 3-Nitroty
Page 55 and 56:
36 Boerner et al. containing Tris,
Page 57 and 58:
38 Boerner et al. 2.2. Nitric Acid
Page 59 and 60:
40 Boerner et al. 8. Böhlen, P., S
Page 61 and 62:
42 Aitken and Learmonth 1.2. Measur
Page 63 and 64:
44 Aitken and Learmonth References
Page 65 and 66:
46 Friedrich et al. 2. Materials 2.
Page 67 and 68:
48 Friedrich et al. Fig. 1. Differe
Page 69 and 70:
50 Friedrich et al. References 1. S
Page 71 and 72:
52 Root and Wang Fig. 1. Representa
Page 73 and 74:
54 Root and Wang 4. Kinetic silver
Page 75 and 76:
Gel Electrophoresis of Proteins 57
Page 77 and 78:
Gel Electrophoresis of Proteins 59
Page 79 and 80:
SDS-PAGE 61 11 SDS Polyacrylamide G
Page 81 and 82:
SDS-PAGE 63 their own rates. A more
Page 83 and 84:
SDS-PAGE 65 7. To ensure that the g
Page 85 and 86:
SDS-PAGE 67 close to the dye, and t
Page 87 and 88:
70 Walker 2. Buffers: a. 1.875 M Tr
Page 89 and 90:
72 Walker Fig. 2. Diagrammatic repr
Page 91 and 92:
74 Judd 2. Materials 2.1. Equipment
Page 93 and 94:
76 Judd ber with anode buffer. Remo
Page 95 and 96:
78 Judd 4. Run the gel as described
Page 97 and 98:
SDecS-PAGE of Nucleic Acid Binding
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
CAT Gel Electrophoresis 87 15 Cetyl
Page 105 and 106:
CAT Gel Electrophoresis 89 Fig. 1.
Page 107 and 108:
CAT Gel Electrophoresis 91 3. CAT s
Page 109 and 110:
CAT Gel Electrophoresis 93 Table 1
Page 111 and 112:
CAT Gel Electrophoresis 95 the tank
Page 113 and 114:
CAT Gel Electrophoresis 97 may be a
Page 115 and 116:
CAT Gel Electrophoresis 99 enzyme p
Page 117 and 118:
CAT Gel Electrophoresis 101 20. Rey
Page 119 and 120:
104 Waterborg Fig. 1. Histones of t
Page 121 and 122:
106 Waterborg 9. Riboflavin-5'-phos
Page 123 and 124:
108 Waterborg 25. Remove the bottom
Page 125 and 126:
110 Waterborg 10. The amount of pro
Page 127 and 128:
AUT Gel for Histones 113 17 Acid-Ur
Page 129 and 130:
AUT Gel for Histones 115 Details of
Page 131 and 132:
AUT Gel for Histones 117 Fig. 2. (A
Page 133 and 134:
AUT Gel for Histones 119 13. Add 0.
Page 135 and 136:
AUT Gel for Histones 121 5. The sep
Page 137 and 138:
AUT Gel for Histones 123 15. Waterb
Page 139 and 140:
126 Walker the cost of preparing IE
Page 141 and 142:
128 Walker 4. Notes 1. The sucrose
Page 143 and 144:
Protein Solubility in 2-D Electroph
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Fractionated Extraction 141 20 Prep
Page 155 and 156:
Fractionated Extraction 143 Fig. 1.
Page 157 and 158:
Fractionated Extraction 145 Table 2
Page 159 and 160:
Fractionated Extraction 147 13. Com
Page 161 and 162:
149 SI + II fraction: liver Brain H
Page 163 and 164:
Fractionated Extraction 151 3. Add
Page 165 and 166:
Fractionated Extraction 153 Fig. 2.
Page 167 and 168:
Fractionated Extraction 155 and bra
Page 169 and 170:
Fractionated Extraction 157 ume are
Page 171 and 172:
160 Bizios 2. Materials 2.1. Equipm
Page 173 and 174:
162 Bizios 5. Sample preparation sh
Page 175 and 176:
164 Gravel Fig. 1. Tube Cell Model
Page 177 and 178:
166 Gravel 3. Fill the lower chambe
Page 179 and 180:
168 Gravel lysis solution A (SDS-DT
Page 181 and 182:
170 Gianazza Fig. 1. Structure of t
Page 183 and 184:
172 Gianazza Table 1 pK Values of I
Page 185 and 186:
174 Gianazza Fig. 2. Course of the
Page 187 and 188:
176 Gianazza Table 3 IPG 4-7 and 6-
Page 189 and 190:
178 Gianazza the gel. Depending on
Page 191 and 192:
180 Gianazza 17. Chiari, M., Pagani
Page 193 and 194:
182 Lopez 3. Slide a grommet onto e
Page 195 and 196:
DIGE 185 25 Difference Gel Electrop
Page 197 and 198:
DIGE 187 2.2. IEF Fig 1. The two cy
Page 199 and 200:
DIGE 189 3. Method 3.1. Sample Solu
Page 201 and 202:
DIGE 191 Strips, the instructions t
Page 203 and 204:
DIGE 193 2. Push the strip down unt
Page 205 and 206:
DIGE 195 4. The presence of Pharmal
Page 207 and 208:
Comparing 2-D Gels on the Internet
Page 209 and 210:
Page 211 and 212:
Page 213 and 214:
Page 215 and 216:
Page 217 and 218:
Page 219 and 220:
Page 221 and 222:
Page 223 and 224:
Page 225 and 226:
Immunoblotting of 2-DE Separated Pr
Page 227 and 228:
Page 229 and 230:
Page 231 and 232:
Page 233 and 234:
Page 235 and 236:
Page 237 and 238:
Page 239 and 240:
Page 241 and 242:
232 Springer Fig. 1. Comparison of
Page 243 and 244:
234 Springer Table 1 Recipe for Var
Page 245 and 246:
Quantification of Proteins on Gels
Page 247 and 248:
Page 249 and 250:
Page 251 and 252:
Rapid Staining with Nile Red 243 30
Page 253 and 254:
Rapid Staining with Nile Red 245 Fi
Page 255 and 256:
Rapid Staining with Nile Red 247 11
Page 257 and 258:
Rapid Staining with Nile Red 249 Re
Page 259 and 260:
252 Fernandez-Patron to years witho
Page 261 and 262:
254 Fernandez-Patron Fig. 2. Revers
Page 263 and 264:
256 Fernandez-Patron spectrometry (
Page 265 and 266:
258 Fernandez-Patron 11. Fernandez-
Page 267 and 268:
260 Choi, Hong, and Yoo Table 1 Lin
Page 269 and 270:
262 Choi, Hong, and Yoo Fig. 2. Mec
Page 271 and 272:
Silver Staining of Proteins 265 33
Page 273 and 274:
Silver Staining of Proteins 267 Fig
Page 275 and 276:
Silver Staining of Proteins 269 3.3
Page 277 and 278:
Silver Staining of Proteins 271 12.
Page 279 and 280:
274 Patton plexes for colorimetric
Page 281 and 282:
276 Patton device (CCD) camera. The
Page 283 and 284:
278 Patton 3.2. Luminescent Detecti
Page 285 and 286:
280 Patton 3.5. Luminescent Detecti
Page 287 and 288:
282 Patton EDTA, pH 9.6 or using SY
Page 289 and 290:
284 Patton filter. Proteins stained
Page 291 and 292:
286 Patton 17. Lim, M., Patton, W.,
Page 293 and 294:
288 Dunn variations in silver stain
Page 295 and 296:
290 Dunn Fig. 1. A 2-DE separation
Page 297 and 298:
292 Dunn 11. Stephens, R. E. (1975)
Page 299 and 300:
Gels Using Eosin Y Stain 295 36 Det
Page 301 and 302:
Gels Using Eosin Y Stain 297 3. An
Page 303 and 304:
300 Jenö and Horst and Coomassie B
Page 305 and 306:
302 Jenö and Horst Fig. 1. Side (A
Page 307 and 308:
304 Jenö and Horst BT1 membrane, o
Page 309 and 310:
Autoradiography and Fluorography 30
Page 311 and 312:
Page 313 and 314:
Page 315 and 316:
Page 317 and 318:
Blotting-Electroblotting 315 PART I
Page 319 and 320:
318 Page and Thorpe 4. Filter paper
Page 322 and 323:
Semidry Protein Blotting 321 40 Pro
Page 324 and 325:
Semidry Protein Blotting 323 2. Mem
Page 326 and 327:
Discontinuous buffer systems Tris-g
Page 328 and 329:
327 Table 2 Membranes Used for Elec
Page 330 and 331:
Semidry Protein Blotting 329 Fig. 2
Page 332 and 333:
Semidry Protein Blotting 331 Table
Page 334 and 335:
Semidry Protein Blotting 333 24 h l
Page 336 and 337:
Protein Blotting 335 41 Protein Blo
Page 338 and 339:
Western Blotting of Basic Proteins
Page 340 and 341:
Page 342 and 343:
Page 344 and 345:
344 Wisdom 4. Glutaraldehyde. 5. 50
Page 346 and 347:
346 Wisdom 3. Methods 1. Dissolve 1
Page 348 and 349:
348 Wisdom 3. Dialyze the modified
Page 350 and 351:
350 Wisdom 6. Store the labeled ant
Page 352 and 353:
352 Mao terminus. The ε-amino grou
Page 354 and 355:
354 Mao conjugate with optimal modi
Page 356 and 357:
356 Haugland and You Fig. 1. Struct
Page 358 and 359:
358 Haugland and You Because of its
Page 360 and 361:
360 Haugland and You 5. Dissolve 10
Page 362 and 363:
362 Haugland and You ing with one a
Page 364 and 365:
Preparation of Avidin Conjugates 36
Page 366 and 367:
Page 368 and 369:
Page 370 and 371:
Page 372 and 373:
Page 374 and 375:
Staining with MDPF 375 50 MDPF Stai
Page 376 and 377:
Staining with MDPF 377 transillumin
Page 378 and 379:
Staining with MDPF 379 3. Alba, F.
Page 380 and 381:
382 Root and Wang suspension become
Page 382 and 383:
384 Root and Wang Fig. 1. Schematic
Page 384 and 385:
Blots Using Direct Blue 71 387 52 D
Page 386 and 387:
Blots Using Direct Blue 71 389 3.2.
Page 388 and 389:
Blots Using Direct Blue 71 391 Fig.
Page 390 and 391:
Immunogold 393 53 Protein Staining
Page 392 and 393:
Immunogold 395 Fig. 2. Schematic re
Page 394 and 395:
Immunogold 397 6. AuroProbe BLplus
Page 396 and 397:
Immunogold 399 Fig. 5. Comparison o
Page 398 and 399:
Immunogold 401 Table 2 Effect of Te
Page 400 and 401:
Immunogold 403 15. AuroDye forte is
Page 402 and 403:
Immunoblotting Using Secondary Liga
Page 404 and 405:
Page 406 and 407:
Page 408 and 409:
Page 410 and 411:
Page 412 and 413:
Avidin-or Streptavidin-Biotin 415 5
Page 414 and 415:
Avidin-or Streptavidin-Biotin 417 2
Page 416 and 417:
Avidin-or Streptavidin-Biotin 419 R
Page 418 and 419:
422 Copse and Fowler substrate to a
Page 420 and 421:
424 Copse and Fowler 2. Orbital sha
Page 422 and 423:
426 Copse and Fowler 4. Notes 4.1.
Page 424 and 425:
428 Copse and Fowler 18. DDAO-phosp
Page 426 and 427:
430 Dickinson and Fowler the advant
Page 428 and 429:
432 Dickinson and Fowler 2. Membran
Page 430 and 431:
434 Dickinson and Fowler Fig. 2. (A
Page 432 and 433:
436 Dickinson and Fowler biotinylat
Page 435 and 436:
Reutilization of Western Blots 439
Page 437 and 438:
Page 439 and 440:
Page 441 and 442:
Page 443 and 444:
Page 445 and 446:
Page 447 and 448:
Page 449 and 450:
Carboxymethylation of Cysteine 453
Page 451 and 452:
456 Aitken and Learmonth 11. Microd
Page 453 and 454:
458 Aitken and Learmonth Table 1 El
Page 455 and 456:
460 Aitken and Learmonth 4. Notes 1
Page 457 and 458:
462 Ward Fig. 1. Amino acid sequenc
Page 459 and 460:
Chemical Modifications of Proteins
Page 461 and 462:
Chemical Modifications of Proteins
Page 463 and 464:
470 Tawfik 3. Method 3.1. Nitration
Page 465 and 466:
472 Tawfik preparation by pH-depend
Page 467 and 468:
474 Tawfik 4. Notes 1. The concentr
Page 469 and 470:
476 Tawfik 2. A molar excess of p-h
Page 471 and 472:
478 Tawfik 3. Method 1. Add the gly
Page 473 and 474:
480 Tawfik 3. Method 1. Dissolve th
Page 475 and 476:
482 Tawfik 4. Notes 1. At neutral a
Page 477 and 478:
484 Tawfik 4. Notes 1. Release of t
Page 479 and 480:
486 Smith 6. Equipment includes a n
Page 481 and 482:
488 Smith 3. Although the specifici
Page 483 and 484:
490 Smith those bearing a prolyl re
Page 485 and 486:
Cleavage at Tryptophanyl-x Bonds 49
Page 487 and 488:
Alternative conditions for rapid re
Page 489 and 490:
Cleavage at Tryptophanyl-x Bonds 49
Page 491 and 492:
Cleavage at Aspartyl-X Bonds 499 73
Page 493 and 494:
Cleavage at Aspartyl-X Bonds 501 pr
Page 495 and 496:
Cleavage at Cysteinyl-x Bonds 503 7
Page 497 and 498:
Cleavage at Cysteinyl-x Bonds 505 F
Page 499 and 500:
Cleavage at Asparaginyl-Glycyl Bond
Page 501 and 502:
Cleavage at Asparaginyl-Glycyl Bond
Page 503 and 504:
Enzymatic Digestion 511 76 Enzymati
Page 505 and 506:
Enzymatic Digestion 513 then remove
Page 507 and 508:
Enzymatic Digestion 515 Fig. 1. In-
Page 509 and 510:
Enzymatic Digestion 517 In the case
Page 511 and 512:
Enzymatic Digestion 519 Another app
Page 513 and 514:
Enzymatic Digestion 521 11. Modific
Page 515 and 516:
524 Table 1 Digestion Buffers Recip
Page 517 and 518:
526 Fernandez and Mische Fig. 1. Pe
Page 519 and 520:
528 Fernandez and Mische 3. Excise
Page 521 and 522:
530 Fernandez and Mische 3. The lar
Page 523 and 524:
532 Fernandez and Mische membrane a
Page 525 and 526:
534 Stone and Williams 2. Materials
Page 527 and 528:
536 Stone and Williams In those few
Page 529 and 530:
538 Stone and Williams (while maint
Page 531 and 532:
540 Stone and Williams Biochemistry
Page 533 and 534:
2-D TLE-TLC Mapping 543 79 Peptide
Page 535 and 536:
2-D TLE-TLC Mapping 545 12. 0.25% N
Page 537 and 538:
2-D TLE-TLC Mapping 547 3. Incubate
Page 539 and 540:
2-D TLE-TLC Mapping 549 Table 1 Cle
Page 541 and 542:
2-D TLE-TLC Mapping 551 Fig. 1. Exa
Page 543 and 544:
SDS-PAGE Mapping 553 80 Peptide Map
Page 545 and 546:
SDS-PAGE Mapping 555 3. Overlay sam
Page 547 and 548:
SDS-PAGE Mapping 557 Fig. 1. Exampl
Page 549 and 550:
560 Judd Fig. 1. Example of peptide
Page 551 and 552:
Protein Hydrolysates 563 82 Product
Page 553 and 554:
Protein Hydrolysates 565 2. Pronase
Page 555 and 556:
Amino Acid Analysis with Marfey’s
Page 557 and 558:
Page 559 and 560:
Page 561 and 562:
HPSEC 573 84 Molecular Weight Estim
Page 563 and 564:
HPSEC 575 Table 1 Protein Standards
Page 565 and 566:
HPSEC 577 Fig. 2. Plot of K d vs lo
Page 567 and 568:
HPSEC 579 with care (see suppliers
Page 569 and 570:
582 Aitken larly good resolution de
Page 571 and 572:
Disulfide-Linked Peptide Detection
Page 573 and 574:
Disulfide-Linked Peptide Detection
Page 575 and 576:
Disulfide Bridges 589 87 Diagonal E
Page 577 and 578:
Disulfide Bridges 591 3. Spot the s
Page 579 and 580:
Disulfide Bridges 593 2. The moveme
Page 581 and 582:
596 Aitken and Learmonth 6. Finally
Page 583 and 584:
598 Aitken and Learmonth Fig. 1. (A
Page 585 and 586:
600 Aitken and Learmonth 2.4. Elect
Page 587 and 588:
602 Aitken and Learmonth 6. Takahas
Page 589 and 590:
604 Colyer of the protein of intere
Page 591 and 592:
606 Colyer 8. After 16 h of exposur
Page 593 and 594:
608 Colyer stoichiometry, since it
Page 595 and 596:
610 Bonenfant, Mini, and Jenö indi
Page 597 and 598:
612 Bonenfant, Mini, and Jenö for
Page 599 and 600:
614 Bonenfant, Mini, and Jenö incl
Page 601 and 602:
616 Bonenfant, Mini, and Jenö Fig.
Page 603 and 604:
618 Bonenfant, Mini, and Jenö Fig.
Page 605 and 606:
620 Bonenfant, Mini, and Jenö up t
Page 607 and 608:
622 Bonenfant, Mini, and Jenö 5. L
Page 609 and 610:
624 Weber and McFadden Fig. 1. Sche
Page 611 and 612:
626 Weber and McFadden 4. Using a p
Page 613 and 614:
628 Weber and McFadden Fig. 2. Comp
Page 615 and 616:
630 Weber and McFadden Fig. 4. Coom
Page 617 and 618:
Protein Palmitoylation 633 93 Analy
Page 619 and 620:
Protein Palmitoylation 635 Fig. 1.
Page 621 and 622:
Protein Palmitoylation 637 1. Fix p
Page 623 and 624:
Protein Palmitoylation 639 7. Mumby
Page 625 and 626:
Fig. 1. Structure of the natural an
Page 627 and 628:
644 Corsini 8. Simvastatin in its l
Page 629 and 630:
646 Corsini Fig. 3. Schematic for t
Page 631 and 632:
648 Corsini Table 1 Mevalonate, Pre
Page 633 and 634:
650 Corsini Fig. 5. Metabolic label
Page 635 and 636:
652 Corsini Fig. 7. Two-dimensional
Page 637 and 638:
654 Corsini 7. When prenylated prot
Page 639 and 640:
656 Corsini 24. Danesi, R., McLella
Page 641 and 642:
658 Andres et al. Fig. 1. Proposed
Page 643 and 644: 660 Andres et al. Fig. 2. SDS-PAGE
Page 645 and 646: 662 Andres et al. Fig. 4. Chromatog
Page 647 and 648: 664 Andres et al. Fig. 6. Specific
Page 649 and 650: 666 Andres et al. 10. Residual orga
Page 651 and 652: 668 Andres et al. 2. The metabolica
Page 653 and 654: 670 Andres et al. 3. Clarke, S. (19
Page 655 and 656: Phosphopeptide Mapping 673 96 2-D P
Page 657 and 658: Phosphopeptide Mapping 675 3. Add 1
Page 659 and 660: Phosphopeptide Mapping 677 Fig. 1.
Page 661 and 662: Phosphopeptide Mapping 679 until th
Page 663 and 664: Protein Mutations by MS 681 97 Dete
Page 665 and 666: Protein Mutations by MS 683 protein
Page 667 and 668: Protein Mutations by MS 685 Fig. 2.
Page 673 and 674: Protein Mutations by MS 691 Fig. 10
Page 675 and 676: Nanoelectrospray MS/MS for Peptide
Page 693: MALDI-MS for Protein Identification
Page 697 and 698: MALDI-MS for Protein Identification
Page 715 and 716: Protein Ladder Sequencing 733 100 P
Page 717 and 718: Protein Ladder Sequencing 735 Prote
Page 719 and 720: Protein Ladder Sequencing 737 3. Ex
Page 721 and 722: Protein Ladder Sequencing 739 2. Wa
Page 723 and 724: 742 Hennig sequences (see Subheadin
Page 725 and 726: 744 Hennig In the age of genomics,
Page 727 and 728: 746 Hennig last defined (last in th
Page 729 and 730: 748 Spencer and Davie Fig. 1. Two-d
Page 731 and 732: 750 Spencer and Davie 4. Notes 1. T
Page 733 and 734: Proteins Cross-linked to DNA by For
Page 739 and 740: Glycoprotein Detection 761 104 Dete
Page 741 and 742: Glycoprotein Detection 763 Schiff
Page 743 and 744: Glycoprotein Detection 765 3.1.1. G
Page 745 and 746:
Glycoprotein Detection 767 Table 1
Page 747 and 748:
Glycoprotein Detection 769 5. Resus
Page 749 and 750:
Glycoprotein Detection 771 Fig. 1.
Page 751 and 752:
Detection of Glycosylated Proteins
Page 753 and 754:
Page 755 and 756:
Page 757 and 758:
780 Gravel Fig. 1. Glycoprotein blo
Page 759 and 760:
782 Gravel Fig. 3. Glycoprotein blo
Page 761 and 762:
784 Table 1 Glycans N-Glycosidicall
Page 763 and 764:
786 Table 3 Specificity of Lectins
Page 765 and 766:
788 Table 3 Specificity of Lectins
Page 767 and 768:
790 Gravel dimethylformamide. These
Page 769 and 770:
792 Gravel 3. Montreuil, J., Bouque
Page 771 and 772:
794 Gravel
Page 773 and 774:
796 Goodarzi, Fotinopoulou, Turner
Page 775 and 776:
Page 777 and 778:
Page 779 and 780:
Page 781 and 782:
804 Hounsell, Davies, and Smith 2.
Page 783 and 784:
Page 785 and 786:
Monosaccharide Analysis by GC 809 1
Page 787 and 788:
Linkage and Substitution Patterns 8
Page 789 and 790:
Linkage and Substitution Patterns 8
Page 791 and 792:
Page 793 and 794:
Page 795 and 796:
820 Hounsell, Davis, and Smith 6. T
Page 797 and 798:
Page 799 and 800:
824 Mizuochi and Hounsell 3. Method
Page 801 and 802:
826 Mizuochi and Hounsell Reference
Page 803 and 804:
Page 805 and 806:
Page 807 and 808:
Page 809 and 810:
834 Weitzhandler et al. alditols (1
Page 811 and 812:
836 Weitzhandler et al. Fig. 1. HPA
Page 813 and 814:
838 Weitzhandler et al. 2. Add 0.1
Page 815 and 816:
Microassay of Protein Glycosylation
Page 817 and 818:
Page 819 and 820:
Page 821 and 822:
Page 823 and 824:
Page 825 and 826:
Carbohydrate Electrophoresis 851 12
Page 827 and 828:
Carbohydrate Electrophoresis 853 2.
Page 829 and 830:
Carbohydrate Electrophoresis 855 Pl
Page 831 and 832:
Page 833 and 834:
Carbohydrate Electrophoresis 859 Fi
Page 835 and 836:
Carbohydrate Electrophoresis 861 wa
Page 837 and 838:
Page 839 and 840:
866 Merry blly less expensive than
Page 841 and 842:
868 Merry 8. Whatman no. 3 chromato
Page 843 and 844:
870 Merry 4. Dialyze for a minimum
Page 845 and 846:
872 Merry 9. Leave for 5 min and un
Page 847 and 848:
874 Merry 2. Column: Reverse-phase
Page 849 and 850:
Table 1 Incubation Conditions for E
Page 851 and 852:
878 Merry 3.7. Exoglycosidase Diges
Page 853 and 854:
880 Merry Fig. 6. Example of exogly
Page 855 and 856:
882 Merry 17. Rice, K. G., Takahash
Page 857 and 858:
Glycoprofiling and SPR 885 124 Glyc
Page 859 and 860:
Glycoprofiling and SPR 887 2. Mater
Page 861 and 862:
Glycoprofiling and SPR 889 Fig. 3.
Page 863 and 864:
Glycoprofiling and SPR 891 α2-3 Ne
Page 865 and 866:
894 Turnbull Fig. 1. Basic principl
Page 867 and 868:
896 Turnbull 13. Enzyme buffer (0.2
Page 869 and 870:
898 Turnbull Table 1 Exoenzymes for
Page 871 and 872:
900 Turnbull Fig. 3. IGS of a hepar
Page 873 and 874:
902 Turnbull 4. Notes 1. Using larg
Page 875 and 876:
904 Turnbull 20. Rice, K., Rottink,
Page 877 and 878:
906 Hooker and James 3. High-perfor
Page 879 and 880:
908 Hooker and James Fig. 1. Whole
Page 881 and 882:
910 Hooker and James at individual
Page 883 and 884:
912 Hooker and James Fig. 4. Sialyl
Page 885 and 886:
914 Hooker and James 16. Ashton, D.
Page 887 and 888:
Lectin Affinity Chromatography 917
Page 889 and 890:
Page 891 and 892:
Page 893 and 894:
Page 895 and 896:
Page 897 and 898:
Page 899 and 900:
Page 901 and 902:
Page 903 and 904:
Antibody Production 935 128 Antibod
Page 905 and 906:
Antibody Production 937 1.1. Donor
Page 907 and 908:
Antibody Production 939 2. Material
Page 909 and 910:
Production of Anitbodies Using Prot
Page 911 and 912:
Production of Anitbodies Using Prot
Page 913 and 914:
Production of Polyclonal Antibodies
Page 915 and 916:
Page 917 and 918:
Page 919 and 920:
Page 921 and 922:
Peptide Conjugation and Immunizatio
Page 923 and 924:
Page 925 and 926:
Page 927 and 928:
Page 929 and 930:
Page 931 and 932:
964 Bailey is oxidized by chloramin
Page 933 and 934:
The Lactoperoxidase Method 967 133
Page 935 and 936:
The Bolton and Hunter Method 969 13
Page 937 and 938:
Radioiodination Using IODO-GEN 971
Page 939 and 940:
Page 941 and 942:
Page 943 and 944:
Page 945 and 946:
980 Bailey 3. Specific antiseum to
Page 947 and 948:
982 Bailey Amount of radioactivity
Page 949 and 950:
984 Page and Thorpe 2. Centrifuge s
Page 951 and 952:
DEAE-Sepharose Chromatography 987 1
Page 953 and 954:
IgG Purification with Ion-Exchange
Page 955 and 956:
PEG Precipitation 991 141 Purificat
Page 957 and 958:
994 Page and Thorpe 2. Materials 1.
Page 959 and 960:
996 Dolman and Thorpe Fig. 1. Typic
Page 961 and 962:
Antigen-Ligand Columns 999 144 Puri
Page 963 and 964:
Antigen-Ligand Columns 1001 4. When
Page 965 and 966:
1004 Page and Thorpe 7. Filter and
Page 967 and 968:
1006 Page and Thorpe Fig. 1. SDS-PA
Page 969 and 970:
Purification of IgY from Chicken Eg
Page 971 and 972:
Purification of IgY from Chicken Eg
Page 973 and 974:
1014 Fassina et al. limit the use o
Page 975 and 976:
1016 Fassina et al. 2. Materials Ta
Page 977 and 978:
1018 Fassina et al. 10. Filter the
Page 979 and 980:
1020 Fassina et al. 1. Dilute sampl
Page 981 and 982:
1022 Fassina et al. analysis indica
Page 983 and 984:
1024 Fassina et al. 6. Khayam-Bashi
Page 985 and 986:
1026 Hammerl et al. down, with the
Page 987 and 988:
1028 Hammerl et al. 1. Clean glass
Page 989 and 990:
1030 Hammerl et al. Fig. 1. Experim
Page 991 and 992:
1032 Hammerl et al. can “corrode
Page 993 and 994:
Single-Chain Antibodies 1035 150 Ba
Page 995 and 996:
Single-Chain Antibodies 1037 ing th
Page 997 and 998:
Single-Chain Antibodies 1039 6. Mag
Page 999 and 1000:
Single-Chain Antibodies 1041 XL1-Bl
Page 1001 and 1002:
Single-Chain Antibodies 1043 fore,
Page 1003 and 1004:
Single-Chain Antibodies 1045 16. Ho
Page 1005 and 1006:
Enzymatic Digestion of MAbs 1047 15
Page 1007 and 1008:
Enzymatic Digestion of MAbs 1049 2.
Page 1009 and 1010:
Enzymatic Digestion of MAbs 1051 2.
Page 1011 and 1012:
Making Bispecific Antibodies 1053 1
Page 1013 and 1014:
Page 1015 and 1016:
Making Bispecific Antibodies 1057 F
Page 1017 and 1018:
Phage Display 1059 153 Phage Displa
Page 1019 and 1020:
Phage Display 1061 5000, 1 mL of 2-
Page 1021 and 1022:
Phage Display 1063 11. Agarose top:
Page 1023 and 1024:
Phage Display 1065 3.1.3.2. AFFINIT
Page 1025 and 1026:
Phage Display 1067 8. Shake vigorou
Page 1027 and 1028:
Phage Display 1069 4. Notes 1. Fila
Page 1029 and 1030:
Phage Display 1071 9. Sengupta J.,
Page 1031 and 1032:
Selection by Panning 1073 154 Scree
Page 1033 and 1034:
Selection by Panning 1075 Fig. 1. F
Page 1035 and 1036:
Selection by Panning 1077 12. Centr
Page 1037 and 1038:
Selection by Panning 1079 4. Notes
Page 1039 and 1040:
Selection by Panning 1081 33. The
Page 1041 and 1042:
Antigen Measurement Using ELISA 108
Page 1043 and 1044:
Page 1045 and 1046:
Page 1047 and 1048:
Enhanced Chemiluminescence 1089 156
Page 1049 and 1050:
Enhanced Chemiluminescence 1091 Tab
Page 1051 and 1052:
Enhanced Chemiluminescence 1093 is
Page 1053 and 1054:
Enhanced Chemiluminescence 1095 lig
Page 1055 and 1056:
Immunoprecipitation 1097 157 Immuno
Page 1057 and 1058:
Immunoprecipitation 1099 Table 2 Pr
Page 1059 and 1060:
Immunoprecipitation 1101 oligosacch
Page 1061 and 1062:
Immunoprecipitation 1103 6. Very ge
Page 1063 and 1064:
Immunoprecipitation 1105 2. Dry the
Page 1065 and 1066:
Short Chapter Title 1107 PART VIII
Page 1067 and 1068:
1110 Page and Thorpe final quantity
Page 1069 and 1070:
1112 Page and Thorpe 2. Materials 1
Page 1071 and 1072:
Page 1073 and 1074:
161 From: The Protein Protocols Han
Page 1075 and 1076:
162 From: The Protein Protocols Han
Page 1077 and 1078:
Affinity Purification of Monoclonal
Page 1079 and 1080:
Page 1081 and 1082:
Page 1083 and 1084:
Page 1085 and 1086:
An Efficient Method for MAb Product
Page 1087 and 1088:
Page 1089 and 1090:
Page 1091 and 1092:
Page 1093 and 1094:
Page 1095 and 1096:
Index 1139 Index A Absorbance (UV),
Page 1097 and 1098:
Index 1141 Electrophoresis of antib
Page 1099 and 1100:
Index 1143 of glycoproteins, 905-91
Page 1101 and 1102:
Index 1145 India ink, 338 lectin st
Page 1103:
The Protein Protocols Handbook Seco
show all

Protein Protocols Protein Protocols

Create successful ePaper yourself

Delete template?

Save as template?