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354 Mao conjugate with optimal modification. Equilibrate and load the column with 10 mM phosphate buffer, pH 8.0. Wash the column with equilibrating buffer and elute with the same buffer containing 100 mM NaCl (first) and 250 mM NaCl (last). Measure the F/P ratios of each fraction, and select the appropriate fractions. 6. Alexa fluorochromes are available only as a protein labeling kit from Molecular Probes, Inc. (Eugene, Oregon, USA; www.probes.com). The reactive dye has a succinimidyl ester moiety that reacts with primary amines of proteins. The conjugation steps are similar to those for fluorescein isothiocyanate. References 1. Coons, A. H., Creech, H. J., and Jones, R. N. (1941) Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47, 200–202. 2. Coons, A. H., Leduc, E. H., and Connolly, J. M. (1955) Studies on antibody production I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J. Exp. Med. 102, 49–60. 3. Mullins, J. M. (1999) Overview of fluorochromes, in Methods in Molecular Biology, vol. 115 (Javois, L. C., ed), Humana Press, USA, pp. 97–105. 4. Titus, J. A., Haugland, R., Sharrow, S. O., and Segal, D. M. (1982) Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies. J. Immunol. Meth. 50, 193–204. 5. Oi, V. T., Glazer, A. N., and Stryer, L. (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. J. Cell Biol. 93, 981–986. 6. Bochner, B. S., McKelvey, A. A., Schleimer, R. P., Hildreth, J. E., and MacGlashan, D. W., Jr. (1989) Flow cytometric methods for the analysis of human basophil surface antigens and viability. J. Immunol. Meth. 125, 265–271. 7. Panchuk-Voloshina, N., Haugland, R. P., Bishop-Stewart, J., Bhalgat, M. K., Millard, P. J., Mao, F., Leung, W. Y., and Haugland, R. P. (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188. 8. Schreiber, A. B. and Haimovich, J. (1983) Quantitative fluorometric assay for detection and characterization of Fc receptors. Meth. Enzymol. 93, 147–155.
Coupling of Abs with Biotin 355 48 Coupling of Antibodies with Biotin Rosaria P. Haugland and Wendy W. You 1. Introduction The avidin–biotin bond is the strongest known biological interaction between a ligand and a protein (K d = 1.3 × 10 –15 M at pH 5) (1). The affinity is so high that the avidin–biotin complex is extremely resistant to any type of denaturing agent (2). Biotin (Fig. 1) is a small, hydrophobic molecule that functions as a coenzyme of carboxylases (3). It is present in all living cells. Avidin is a tetrameric glycoprotein of 66,000–68,000 mol wt, found in egg albumin and in avian tissues. The interaction between avidin and biotin occurs rapidly, and the stability of the complex has prompted its use for in situ attachment of labels in a broad variety of applications, including immunoassays, DNA hybridization (4–6), and localization of antigens in cells and tissues (7). Avidin has an isoelectric point of 10.5. Because of its positively charged residues and its oligosaccharide component, consisting mostly of mannose and glucosamine (8), avidin can interact nonspecifically with negative charges on cell surfaces and nucleic acids, or with membrane sugar receptors. At times, this causes background problems in histochemical and cytochemical applications. Streptavidin, a near-neutral, biotin binding protein (9) isolated from the culture medium of Streptomyces avidinii, is a tetrameric nonglycosylated analog of avidin with a mol wt of about 60,000. Like avidin, each molecule of streptavidin binds four molecules of biotin, with a similar dissociation constant. The two proteins have about 33% sequence homology, and tryptophan residues seem to be involved in their biotin binding sites (10,11). In general, streptavidin gives less background problems than avidin. This protein, however, contains a tripeptide sequence Arg-Tyr-Asp (RYD) that apparently mimics the binding sequence of fibronectin Arg-Gly-Asp (RGD), a universal recognition domain of the extracellular matrix that specifically promotes cell adhesion. Consequently, the streptavidin–cell-surface interaction causes high background in certain applications (12). As an alternative to both avidin and streptavidin, a chemically modified avidin, NeutrAvidin (NeutrAvidin is a trademark of Pierce Chemical Company, Rockford, IL) and its conjugates with enzymes or fluorescent probes are available from both Molecular Probes and Pierce. NeutrAvidin consists of chemically deglycosylated avidin, which has been modified to reduce the isoelectric point to a neutral value, without loss of its biotin binding properties and without significant change in the lysines avail- From: The <strong>Protein</strong> <strong>Protocols</strong> Handbook, 2nd Edition Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ 355
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The Protein Protocols Handbook SECO
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The Protein Protocols Handbook SECO
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Preface The Protein Protocols Handb
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viii Contents 14 Identification of
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x Contents 47 Conjugation of Fluoro
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xii Contents 80 Peptide Mapping by
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xiv Contents 112 Sialic Acid Analys
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xvi Contents 145 Purification of Ig
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Contributors THOMAS E. ADRIAN • D
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Contributors xxi RUTH R. FRENCH •
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Contributors xxiii STEFANIE A. NELS
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UV Absorption 1 PART I QUANTITATION
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4 Aitken and Learmonth 2. Materials
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6 Aitken and Learmonth References 1
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8 Waterborg 3. Method 1. To 0.1 mL
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BCA for Protein Quantitation 11 3 T
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BCA for Protein Quantitation 13 Tab
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The Bradford Method 15 4 The Bradfo
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The Bradford Method 17 Fig. 1. Vari
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The Bradford Method 19 Table 2 Comp
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The Bradford Method 21 13. Kirazov,
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24 Akins and Tuan passes. The side
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26 Akins and Tuan Fig. 2. Effect of
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28 Akins and Tuan protein concentra
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30 Akins and Tuan energy. Too much
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32 Boerner et al. Several approache
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34 Boerner et al. Fig. 2. 3-Nitroty
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36 Boerner et al. containing Tris,
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38 Boerner et al. 2.2. Nitric Acid
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40 Boerner et al. 8. Böhlen, P., S
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42 Aitken and Learmonth 1.2. Measur
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44 Aitken and Learmonth References
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46 Friedrich et al. 2. Materials 2.
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48 Friedrich et al. Fig. 1. Differe
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50 Friedrich et al. References 1. S
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52 Root and Wang Fig. 1. Representa
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54 Root and Wang 4. Kinetic silver
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Gel Electrophoresis of Proteins 57
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Gel Electrophoresis of Proteins 59
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SDS-PAGE 61 11 SDS Polyacrylamide G
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SDS-PAGE 63 their own rates. A more
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SDS-PAGE 65 7. To ensure that the g
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SDS-PAGE 67 close to the dye, and t
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70 Walker 2. Buffers: a. 1.875 M Tr
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72 Walker Fig. 2. Diagrammatic repr
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74 Judd 2. Materials 2.1. Equipment
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76 Judd ber with anode buffer. Remo
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78 Judd 4. Run the gel as described
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SDecS-PAGE of Nucleic Acid Binding
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CAT Gel Electrophoresis 87 15 Cetyl
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CAT Gel Electrophoresis 89 Fig. 1.
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CAT Gel Electrophoresis 91 3. CAT s
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CAT Gel Electrophoresis 93 Table 1
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CAT Gel Electrophoresis 95 the tank
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CAT Gel Electrophoresis 97 may be a
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CAT Gel Electrophoresis 99 enzyme p
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CAT Gel Electrophoresis 101 20. Rey
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104 Waterborg Fig. 1. Histones of t
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106 Waterborg 9. Riboflavin-5'-phos
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108 Waterborg 25. Remove the bottom
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110 Waterborg 10. The amount of pro
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AUT Gel for Histones 113 17 Acid-Ur
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AUT Gel for Histones 115 Details of
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AUT Gel for Histones 117 Fig. 2. (A
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AUT Gel for Histones 119 13. Add 0.
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AUT Gel for Histones 121 5. The sep
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AUT Gel for Histones 123 15. Waterb
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126 Walker the cost of preparing IE
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128 Walker 4. Notes 1. The sucrose
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Protein Solubility in 2-D Electroph
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Fractionated Extraction 141 20 Prep
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Fractionated Extraction 143 Fig. 1.
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Fractionated Extraction 145 Table 2
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Fractionated Extraction 147 13. Com
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149 SI + II fraction: liver Brain H
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Fractionated Extraction 151 3. Add
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Fractionated Extraction 153 Fig. 2.
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Fractionated Extraction 155 and bra
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Fractionated Extraction 157 ume are
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160 Bizios 2. Materials 2.1. Equipm
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162 Bizios 5. Sample preparation sh
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164 Gravel Fig. 1. Tube Cell Model
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166 Gravel 3. Fill the lower chambe
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168 Gravel lysis solution A (SDS-DT
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170 Gianazza Fig. 1. Structure of t
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172 Gianazza Table 1 pK Values of I
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174 Gianazza Fig. 2. Course of the
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176 Gianazza Table 3 IPG 4-7 and 6-
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178 Gianazza the gel. Depending on
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180 Gianazza 17. Chiari, M., Pagani
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182 Lopez 3. Slide a grommet onto e
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DIGE 185 25 Difference Gel Electrop
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DIGE 187 2.2. IEF Fig 1. The two cy
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DIGE 189 3. Method 3.1. Sample Solu
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DIGE 191 Strips, the instructions t
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DIGE 193 2. Push the strip down unt
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DIGE 195 4. The presence of Pharmal
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Comparing 2-D Gels on the Internet
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Immunoblotting of 2-DE Separated Pr
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232 Springer Fig. 1. Comparison of
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234 Springer Table 1 Recipe for Var
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Quantification of Proteins on Gels
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Rapid Staining with Nile Red 243 30
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Rapid Staining with Nile Red 245 Fi
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Rapid Staining with Nile Red 247 11
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Rapid Staining with Nile Red 249 Re
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252 Fernandez-Patron to years witho
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254 Fernandez-Patron Fig. 2. Revers
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256 Fernandez-Patron spectrometry (
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258 Fernandez-Patron 11. Fernandez-
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260 Choi, Hong, and Yoo Table 1 Lin
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262 Choi, Hong, and Yoo Fig. 2. Mec
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Silver Staining of Proteins 265 33
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Silver Staining of Proteins 267 Fig
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Silver Staining of Proteins 269 3.3
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Silver Staining of Proteins 271 12.
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274 Patton plexes for colorimetric
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276 Patton device (CCD) camera. The
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278 Patton 3.2. Luminescent Detecti
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280 Patton 3.5. Luminescent Detecti
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282 Patton EDTA, pH 9.6 or using SY
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284 Patton filter. Proteins stained
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286 Patton 17. Lim, M., Patton, W.,
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288 Dunn variations in silver stain
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290 Dunn Fig. 1. A 2-DE separation
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292 Dunn 11. Stephens, R. E. (1975)
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Gels Using Eosin Y Stain 295 36 Det
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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 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 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 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 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
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Immunoblotting Using Secondary Liga
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Avidin-or Streptavidin-Biotin 415 5
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Avidin-or Streptavidin-Biotin 417 2
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Avidin-or Streptavidin-Biotin 419 R
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422 Copse and Fowler substrate to a
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424 Copse and Fowler 2. Orbital sha
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426 Copse and Fowler 4. Notes 4.1.
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428 Copse and Fowler 18. DDAO-phosp
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430 Dickinson and Fowler the advant
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432 Dickinson and Fowler 2. Membran
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434 Dickinson and Fowler Fig. 2. (A
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436 Dickinson and Fowler biotinylat
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Reutilization of Western Blots 439
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Carboxymethylation of Cysteine 453
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456 Aitken and Learmonth 11. Microd
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458 Aitken and Learmonth Table 1 El
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460 Aitken and Learmonth 4. Notes 1
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462 Ward Fig. 1. Amino acid sequenc
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Chemical Modifications of Proteins
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Chemical Modifications of Proteins
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470 Tawfik 3. Method 3.1. Nitration
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472 Tawfik preparation by pH-depend
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474 Tawfik 4. Notes 1. The concentr
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476 Tawfik 2. A molar excess of p-h
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478 Tawfik 3. Method 1. Add the gly
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480 Tawfik 3. Method 1. Dissolve th
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482 Tawfik 4. Notes 1. At neutral a
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484 Tawfik 4. Notes 1. Release of t
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486 Smith 6. Equipment includes a n
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488 Smith 3. Although the specifici
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490 Smith those bearing a prolyl re
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Cleavage at Tryptophanyl-x Bonds 49
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Alternative conditions for rapid re
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Cleavage at Tryptophanyl-x Bonds 49
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Cleavage at Aspartyl-X Bonds 499 73
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Cleavage at Aspartyl-X Bonds 501 pr
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Cleavage at Cysteinyl-x Bonds 503 7
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Cleavage at Cysteinyl-x Bonds 505 F
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Cleavage at Asparaginyl-Glycyl Bond
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Cleavage at Asparaginyl-Glycyl Bond
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Enzymatic Digestion 511 76 Enzymati
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Enzymatic Digestion 513 then remove
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Enzymatic Digestion 515 Fig. 1. In-
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Enzymatic Digestion 517 In the case
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Enzymatic Digestion 519 Another app
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Enzymatic Digestion 521 11. Modific
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524 Table 1 Digestion Buffers Recip
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526 Fernandez and Mische Fig. 1. Pe
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528 Fernandez and Mische 3. Excise
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530 Fernandez and Mische 3. The lar
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532 Fernandez and Mische membrane a
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534 Stone and Williams 2. Materials
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536 Stone and Williams In those few
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538 Stone and Williams (while maint
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540 Stone and Williams Biochemistry
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2-D TLE-TLC Mapping 543 79 Peptide
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2-D TLE-TLC Mapping 545 12. 0.25% N
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2-D TLE-TLC Mapping 547 3. Incubate
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2-D TLE-TLC Mapping 549 Table 1 Cle
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2-D TLE-TLC Mapping 551 Fig. 1. Exa
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SDS-PAGE Mapping 553 80 Peptide Map
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SDS-PAGE Mapping 555 3. Overlay sam
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SDS-PAGE Mapping 557 Fig. 1. Exampl
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560 Judd Fig. 1. Example of peptide
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Protein Hydrolysates 563 82 Product
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Protein Hydrolysates 565 2. Pronase
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Amino Acid Analysis with Marfey’s
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HPSEC 573 84 Molecular Weight Estim
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HPSEC 575 Table 1 Protein Standards
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HPSEC 577 Fig. 2. Plot of K d vs lo
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HPSEC 579 with care (see suppliers
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582 Aitken larly good resolution de
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Disulfide-Linked Peptide Detection
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Disulfide-Linked Peptide Detection
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Disulfide Bridges 589 87 Diagonal E
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Disulfide Bridges 591 3. Spot the s
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Disulfide Bridges 593 2. The moveme
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596 Aitken and Learmonth 6. Finally
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598 Aitken and Learmonth Fig. 1. (A
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600 Aitken and Learmonth 2.4. Elect
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602 Aitken and Learmonth 6. Takahas
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604 Colyer of the protein of intere
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606 Colyer 8. After 16 h of exposur
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608 Colyer stoichiometry, since it
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610 Bonenfant, Mini, and Jenö indi
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612 Bonenfant, Mini, and Jenö for
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614 Bonenfant, Mini, and Jenö incl
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616 Bonenfant, Mini, and Jenö Fig.
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618 Bonenfant, Mini, and Jenö Fig.
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620 Bonenfant, Mini, and Jenö up t
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622 Bonenfant, Mini, and Jenö 5. L
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624 Weber and McFadden Fig. 1. Sche
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626 Weber and McFadden 4. Using a p
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628 Weber and McFadden Fig. 2. Comp
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630 Weber and McFadden Fig. 4. Coom
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Protein Palmitoylation 633 93 Analy
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Protein Palmitoylation 635 Fig. 1.
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Protein Palmitoylation 637 1. Fix p
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Protein Palmitoylation 639 7. Mumby
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Fig. 1. Structure of the natural an
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644 Corsini 8. Simvastatin in its l
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646 Corsini Fig. 3. Schematic for t
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648 Corsini Table 1 Mevalonate, Pre
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650 Corsini Fig. 5. Metabolic label
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652 Corsini Fig. 7. Two-dimensional
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654 Corsini 7. When prenylated prot
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656 Corsini 24. Danesi, R., McLella
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658 Andres et al. Fig. 1. Proposed
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660 Andres et al. Fig. 2. SDS-PAGE
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662 Andres et al. Fig. 4. Chromatog
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664 Andres et al. Fig. 6. Specific
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666 Andres et al. 10. Residual orga
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668 Andres et al. 2. The metabolica
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670 Andres et al. 3. Clarke, S. (19
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Phosphopeptide Mapping 673 96 2-D P
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Phosphopeptide Mapping 675 3. Add 1
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Phosphopeptide Mapping 677 Fig. 1.
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Phosphopeptide Mapping 679 until th
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Protein Mutations by MS 681 97 Dete
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Protein Mutations by MS 683 protein
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Protein Mutations by MS 685 Fig. 2.
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Protein Mutations by MS 691 Fig. 10
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Nanoelectrospray MS/MS for Peptide
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MALDI-MS for Protein Identification
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Protein Ladder Sequencing 733 100 P
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Protein Ladder Sequencing 735 Prote
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Protein Ladder Sequencing 737 3. Ex
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Protein Ladder Sequencing 739 2. Wa
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742 Hennig sequences (see Subheadin
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744 Hennig In the age of genomics,
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746 Hennig last defined (last in th
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748 Spencer and Davie Fig. 1. Two-d
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750 Spencer and Davie 4. Notes 1. T
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Proteins Cross-linked to DNA by For
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Glycoprotein Detection 761 104 Dete
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Glycoprotein Detection 763 Schiff
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Glycoprotein Detection 765 3.1.1. G
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Glycoprotein Detection 767 Table 1
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Glycoprotein Detection 769 5. Resus
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Glycoprotein Detection 771 Fig. 1.
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Detection of Glycosylated Proteins
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780 Gravel Fig. 1. Glycoprotein blo
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782 Gravel Fig. 3. Glycoprotein blo
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784 Table 1 Glycans N-Glycosidicall
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786 Table 3 Specificity of Lectins
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788 Table 3 Specificity of Lectins
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790 Gravel dimethylformamide. These
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792 Gravel 3. Montreuil, J., Bouque
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794 Gravel
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796 Goodarzi, Fotinopoulou, Turner
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Page 777 and 778:
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804 Hounsell, Davies, and Smith 2.
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Monosaccharide Analysis by GC 809 1
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Linkage and Substitution Patterns 8
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Linkage and Substitution Patterns 8
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820 Hounsell, Davis, and Smith 6. T
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824 Mizuochi and Hounsell 3. Method
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826 Mizuochi and Hounsell Reference
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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
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