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Precipitation of IgG 983 137 Purification of IgG by Precipitation with Sodium Sulfate or Ammonium Sulfate Mark Page and Robin Thorpe 1. Introduction Addition of appropriate amounts of salts, such as ammonium or sodium sulfate, causes precipitation of IgG (1) from all mammals, and can be used for serum, plasma, ascites fluid, and hybridoma culture supernatant. Although such IgG is usually contaminated with other proteins, the ease of these precipitation procedures coupled with the high yield of IgG has led to their wide use in producing enriched IgG preparations. They are suitable for many immunochemical procedures, e.g., production of immunoaffinity columns, and as a starting point for further purification. It is not suitable however for conjugating with radiolabels, enzymes, or biotin since the contaminating proteins will also be conjugated, thereby reducing the efficiency of the labeling and the quality of the reagent. The precipitated IgG is usually very stable, and such preparations are ideally suited for long-term storage or distribution and exchange between laboratories. Ammonium sulfate precipitation is the most widely used and adaptable procedure, yielding a 40% pure preparation; sodium sulfate can give a purer preparation for some species, e.g., human and monkey. 2. Materials 2.1. Ammonium Sulfate Precipitation 1. Saturated ammonium sulfate solution: Add excess (NH4) 2SO4 to distilled water (about 950 g to 1 L), and stir overnight at room temperature. Chill at 4°C, and store at this temperature. This solution (in contact with solid salt) is stored at 4°C. 2. PBS: 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4. Store at 4°C. 2.2. Sodium Sulfate Precipitation This requires solid sodium sulfate. 3. Methods 3.1. Ammonium Sulfate Precipitation 1. Prepare saturated ammonium sulfate at least 24 h before the solution is required for fractionation. Store at 4°C. From: The <strong>Protein</strong> <strong>Protocols</strong> Handbook, 2nd Edition Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ 983
984 Page and Thorpe 2. Centrifuge serum or plasma for 20–30 min at 10,000g av at 4°C. Discard the pellet (see Note 1). 3. Cool the serum or plasma to 4°C, and stir slowly. Add saturated ammonium sulfate solution dropwise to produce 35–45% final saturation (see Note 2). Alternatively, add solid ammonium sulfate to give the desired saturation (2.7 g of ammonium sulfate/10 mL of fluid = 45% saturation). Stir at 4°C for 1–4 h or overnight. 4. Centrifuge at 2000–4000g av for 15–20 min at 4°C (alternatively for small volumes of 1–5 mL, microfuge for 1–2 min). Discard the supernatant, and drain the pellet (carefully invert the tube over a paper tissue). 5. Dissolve the precipitate in 10–20% of the original volume in PBS or other buffer by careful mixing with a spatula or drawing repeatedly into a wide-gage Pasteur pipet. When fully dispersed, add more buffer to give 25–50% of the original volume and dialyze against the required buffer (e.g., PBS) at 4°C overnight with two to three buffer changes. Alternatively, the precipitate can be stored at 4 or –20°C if not required immediately. 3.2. Sodium Sulfate Precipitation (see Note 3) 1. Centrifuge the serum or plasma at 10,000gav for 20–30 min. Discard the pellet, warm the serum to 25°C, and stir. 2. Add solid Na2SO4 to produce an 18% w/v solution (i.e., add 1.8 g/10 mL), and stir at 25°C for 30 min to 1 h. 3. Centrifuge at 2000–4000gav for 30 min at 25°C. 4. Discard the supernatant, and drain the pellet. Redissolve in the appropriate buffer as described for ammonium sulfate precipitation (Subheading 3.1., step 5). 4. Notes 1. If lipid contamination is excessive in ascites fluids, thereby compromising the salt precipitation, add silicone dioxide powder (15 mg/ mL) and centrifuge for 20 min at 2000gav (2) before adding the ammonium or sodium salt. 2. The use of 35% ammonium sulfate will produce a pure IgG preparation, but will not precipitate all the IgG present in serum or plasma. Increasing saturation to 45% causes precipitation of nearly all IgG, but this will be contaminated with other proteins, including some albumin. Purification using (NH4) 2SO4 can be improved by repeating the precipitation, but this may cause some denaturation. Precipitation with 45% (NH4) 2SO4 is an ideal starting point for further purification steps, e.g., ion-exchange or affinity chromatography and FPLC purification (see Chapters 139 and 140). 3. Sodium sulfate may be used for precipitation of IgG instead of ammonium sulfate. The advantage of the sodium salt is that a purer preparation of IgG can be obtained, but this must be determined experimentally. The disadvantages are that yield may be reduced depending on the IgG characteristics of the starting material, IgG concentration, and composition. Fractionation must be carried out at a precise temperature (usually 25°C), since the solubility of Na2SO4 is very temperature dependent. Sodium sulfate is usually employed only for the purification of rabbit or human IgG. References 1. Heide, K. and Schwick, H. G. (1978) Salt fractionation of immunoglobulins, in Handbook of Experimental Immunology, 3rd ed. (Weir, D. M., ed.), chap. 7. Blackwell Scientific, Oxford, UK. 2. Neoh, S. H., Gordon, C., Potter, A., and Zola, H. (1986) The purification of mouse MAb from ascitic fluid. J. Immunol. Meth. 91, 231.
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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
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300 Jenö and Horst and Coomassie B
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302 Jenö and Horst Fig. 1. Side (A
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304 Jenö and Horst BT1 membrane, o
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Autoradiography and Fluorography 30
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Blotting-Electroblotting 315 PART I
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318 Page and Thorpe 4. Filter paper
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Semidry Protein Blotting 321 40 Pro
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Semidry Protein Blotting 323 2. Mem
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Discontinuous buffer systems Tris-g
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327 Table 2 Membranes Used for Elec
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Semidry Protein Blotting 329 Fig. 2
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Semidry Protein Blotting 331 Table
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Semidry Protein Blotting 333 24 h l
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Protein Blotting 335 41 Protein Blo
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Western Blotting of Basic Proteins
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344 Wisdom 4. Glutaraldehyde. 5. 50
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346 Wisdom 3. Methods 1. Dissolve 1
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348 Wisdom 3. Dialyze the modified
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350 Wisdom 6. Store the labeled ant
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352 Mao terminus. The ε-amino grou
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354 Mao conjugate with optimal modi
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356 Haugland and You Fig. 1. Struct
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358 Haugland and You Because of its
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360 Haugland and You 5. Dissolve 10
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362 Haugland and You ing with one a
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Preparation of Avidin Conjugates 36
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Staining with MDPF 375 50 MDPF Stai
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Staining with MDPF 377 transillumin
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Staining with MDPF 379 3. Alba, F.
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382 Root and Wang suspension become
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384 Root and Wang Fig. 1. Schematic
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Blots Using Direct Blue 71 387 52 D
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Blots Using Direct Blue 71 389 3.2.
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Blots Using Direct Blue 71 391 Fig.
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Immunogold 393 53 Protein Staining
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Immunogold 395 Fig. 2. Schematic re
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Immunogold 397 6. AuroProbe BLplus
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Immunogold 399 Fig. 5. Comparison o
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Immunogold 401 Table 2 Effect of Te
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Immunogold 403 15. AuroDye forte is
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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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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
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836 Weitzhandler et al. Fig. 1. HPA
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838 Weitzhandler et al. 2. Add 0.1
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Microassay of Protein Glycosylation
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Carbohydrate Electrophoresis 851 12
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Carbohydrate Electrophoresis 853 2.
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Carbohydrate Electrophoresis 855 Pl
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Carbohydrate Electrophoresis 859 Fi
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Carbohydrate Electrophoresis 861 wa
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866 Merry blly less expensive than
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868 Merry 8. Whatman no. 3 chromato
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870 Merry 4. Dialyze for a minimum
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872 Merry 9. Leave for 5 min and un
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874 Merry 2. Column: Reverse-phase
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Table 1 Incubation Conditions for E
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878 Merry 3.7. Exoglycosidase Diges
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880 Merry Fig. 6. Example of exogly
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882 Merry 17. Rice, K. G., Takahash
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Glycoprofiling and SPR 885 124 Glyc
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Glycoprofiling and SPR 887 2. Mater
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Glycoprofiling and SPR 889 Fig. 3.
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Glycoprofiling and SPR 891 α2-3 Ne
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894 Turnbull Fig. 1. Basic principl
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896 Turnbull 13. Enzyme buffer (0.2
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898 Turnbull Table 1 Exoenzymes for
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900 Turnbull Fig. 3. IGS of a hepar
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902 Turnbull 4. Notes 1. Using larg
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904 Turnbull 20. Rice, K., Rottink,
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906 Hooker and James 3. High-perfor
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908 Hooker and James Fig. 1. Whole
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910 Hooker and James at individual
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912 Hooker and James Fig. 4. Sialyl
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914 Hooker and James 16. Ashton, D.
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Lectin Affinity Chromatography 917
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Page 897 and 898: Lectin Affinity Chromatography 927
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 921 and 922: Peptide Conjugation and Immunizatio
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 945 and 946: 980 Bailey 3. Specific antiseum to
Page 947: 982 Bailey Amount of radioactivity
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
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Single-Chain Antibodies 1041 XL1-Bl
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Single-Chain Antibodies 1043 fore,
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Single-Chain Antibodies 1045 16. Ho
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Enzymatic Digestion of MAbs 1047 15
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Enzymatic Digestion of MAbs 1049 2.
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Enzymatic Digestion of MAbs 1051 2.
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Making Bispecific Antibodies 1053 1
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Making Bispecific Antibodies 1057 F
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Phage Display 1059 153 Phage Displa
Page 1019 and 1020:
Phage Display 1061 5000, 1 mL of 2-
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Phage Display 1063 11. Agarose top:
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Phage Display 1065 3.1.3.2. AFFINIT
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Phage Display 1067 8. Shake vigorou
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Phage Display 1069 4. Notes 1. Fila
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Phage Display 1071 9. Sengupta J.,
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Selection by Panning 1073 154 Scree
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Selection by Panning 1075 Fig. 1. F
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Selection by Panning 1077 12. Centr
Page 1037 and 1038:
Selection by Panning 1079 4. Notes
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Selection by Panning 1081 33. The
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Antigen Measurement Using ELISA 108
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Enhanced Chemiluminescence 1089 156
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Enhanced Chemiluminescence 1091 Tab
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Enhanced Chemiluminescence 1093 is
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Enhanced Chemiluminescence 1095 lig
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Immunoprecipitation 1097 157 Immuno
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Immunoprecipitation 1099 Table 2 Pr
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Immunoprecipitation 1101 oligosacch
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Immunoprecipitation 1103 6. Very ge
Page 1063 and 1064:
Immunoprecipitation 1105 2. Dry the
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Short Chapter Title 1107 PART VIII
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1110 Page and Thorpe final quantity
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1112 Page and Thorpe 2. Materials 1
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161 From: The Protein Protocols Han
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162 From: The Protein Protocols Han
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Affinity Purification of Monoclonal
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Page 1083 and 1084:
Page 1085 and 1086:
An Efficient Method for MAb Product
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Index 1139 Index A Absorbance (UV),
Page 1097 and 1098:
Index 1141 Electrophoresis of antib
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Index 1143 of glycoproteins, 905-91
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Index 1145 India ink, 338 lectin st
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The Protein Protocols Handbook Seco
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