List of contributors |
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ix | (4) |
Foreword |
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xiii | (2) |
Introduction |
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xv | (2) |
Abbreviations and symbols |
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xvii | |
PART ONE CELLULAR IMMUNOLOGY TECHNIQUES |
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2 | (112) |
Section 1. Cell preparation |
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1. Peripheral blood mononuclear cell (PBMC) isolation |
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2 | (2) |
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2. Polymorphonuclear cell (PMNC) isolation |
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4 | (2) |
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6 | (4) |
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10 | (2) |
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5. Natural killer (NK) cell preparation |
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12 | (2) |
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6. B cell purification from tonsils |
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14 | (2) |
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7. Positive selection of CD34(+) progenitor cells from cord blood |
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16 | (2) |
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8. Cell purification using magnetic beads |
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18 | (2) |
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9. Isolation and enrichment of human epidermal Langerhans cells |
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20 | (4) |
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10. In vitro generation of dendritic cells |
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24 | (2) |
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11. Generation of T cell clones |
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26 | (4) |
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12. Culture of lympokine activated killer (LAK) cells and tumor infiltrating lymphocytes (TIL) |
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30 | (2) |
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13. Generation of human natural killer (NK) cell clones |
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32 | (4) |
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14. CD40 activation of B lymphocytes |
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36 | (2) |
Section 2. Cell typing |
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15. Membrane immunofluorescence and cocapping |
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38 | (4) |
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16. Immunofluorescence for intracellular antigens |
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42 | (4) |
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17. Detection of FcGamma receptors (FcGammaR) by rosette assay |
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46 | (2) |
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48 | (4) |
Section 3. Cytotoxicity |
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19. Complement-dependent cell cytotoxicity |
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52 | (4) |
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20. Cell-mediated cytotoxicity assay |
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56 | (4) |
Section 4. Cell culture |
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21. Mononuclear cell activation with mitogens |
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60 | (2) |
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22. Measurement of endotoxin level |
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62 | (4) |
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23. Removal of endotoxins |
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66 | (2) |
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68 | (4) |
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72 | (2) |
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26. Cell freezing and thawing |
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74 | (2) |
Section 5. Cell activation |
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76 | (4) |
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28. Protein tyrosine phosphorylation |
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80 | (4) |
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29. Measurement of superoxide release |
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84 | (2) |
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30. Inflammatory mediator release |
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86 | (4) |
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31. Cytokine detection and quantification |
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90 | (2) |
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32. Cytokine mRNA detection |
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92 | (4) |
Section 6. Cell proliferation |
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33. Measurement of cell proliferation by [(3)H]thymidine uptake |
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96 | (4) |
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100 | (2) |
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35. Use of flow cytometry for measurement of T cell proliferation |
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102 | (2) |
Section 7. Apoptosis evaluation |
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36. Internucleosomal DNA fragmentation analysis |
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104 | (2) |
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37. Apoptosis evaluation by flow cytometry: the TUNEL technique |
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106 | (4) |
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38. Apoptosis evaluation by Annexin V-FITC |
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110 | (4) |
PART TWO IMMUNOCHEMICAL TECHNIQUES |
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114 | (130) |
Section 8. Antigen preparation |
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39. Coupling peptides with carrier proteins |
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114 | (2) |
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40. Preparation of immunogens and methods for increasing immunogenicity |
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116 | (2) |
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118 | (2) |
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42. Coupling peptides to liposomes |
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120 | (2) |
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43. Cell extract preparation |
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122 | (2) |
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44. Biosynthetic cell labeling |
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124 | (2) |
Section 9. Generation of polyclonal antibodies |
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126 | (2) |
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46. Bleeding and serum preparation |
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128 | (4) |
Section 10. Generation of monoclonal antibodies and hybridomas |
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47. Monoclonal antibody production: general considerations |
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132 | (2) |
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48. Culture of murine mutant fusion partners |
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134 | (4) |
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49. Cell fusion: obtaining B cell hybridomas |
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138 | (4) |
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50. Selection of positive hybridoma clones and cloning by limiting dilution |
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142 | (4) |
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51. Production of human monoclonal antibodies by Epstein-Barr virus (EBV) transformation |
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146 | (2) |
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52. In vitro cell immunization |
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148 | (4) |
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53. Laboratory-scale antibody production |
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152 | (4) |
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54. Selection of switch variants |
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156 | (4) |
Section 11. Antibody characterization |
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55. Indirect ELISA by antibody capture |
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160 | (4) |
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56. Quantifying antigens by direct competition assay |
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164 | (4) |
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57. Quantifying antigens by ELISA sandwich assay |
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168 | (4) |
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58. Isotype determination of antibodies by sandwich ELISA |
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172 | (4) |
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59. Detection of specific antibodies by double-immunodiffusion |
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176 | (4) |
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60. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) |
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180 | (4) |
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184 | (4) |
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188 | (4) |
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63. Determination of antibody affinity constant (K(aff)) |
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192 | (4) |
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64. Measurement of antibody affinity by solid-phase assay |
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196 | (4) |
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65. Kinetic measurements by surface plasmon resonance (SPR) |
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200 | (4) |
Section 12. Antibody purification |
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66. Ammonium sulfate precipitation |
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204 | (4) |
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67. Antibody purification by caprylic acid precipitation |
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208 | (2) |
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68. Antibody purification by hydroxylapatite chromatography |
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210 | (2) |
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69. Antibody purification by diethyl amino ethyl (DEAE) ion exchange chromatography |
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212 | (2) |
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70. Coupling antibodies to activated Sepharose 4B beads |
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214 | (2) |
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71. Purification of IgG by protein A or protein G affinity chromatography |
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216 | (4) |
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72. Antigen affinity chromatography |
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220 | (2) |
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73. Antigen purification by antibody affinity chromatography |
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222 | (4) |
Section 13. Biochemical antibody engineering |
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74. Antibody labeling with (125)iodine |
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226 | (4) |
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230 | (2) |
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76. Fluorescein isothiocyanate (FITC) coupling |
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232 | (2) |
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77. Coupling enzymes to antibodies |
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234 | (2) |
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78. Preparation of F(ab)(2) from IgG using proteolytic enzymes |
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236 | (4) |
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79. Preparation of Fab from F(ab)(2) |
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240 | (4) |
PART THREE LABORATORY HOSPITAL TECHNIQUES |
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244 | (50) |
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80. Autoantibody detection |
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244 | (2) |
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81. Immune complexes (IC) detection |
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246 | (4) |
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250 | (4) |
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83. Hypergammaglobulinemia and hypogammaglobulinemia detection |
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254 | (4) |
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84. Cryoglobulins (CG) detection and classification |
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258 | (4) |
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85. Monoclonal gammapathies detection by immunoelectrophoresis (IEP) |
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262 | (4) |
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86. Monoclonal gammapathies detection by electroimmunofixation (EIF) |
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266 | (4) |
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87. CD typing by immunofluorescence |
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270 | (4) |
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88. HLA class I serological typing |
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274 | (4) |
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89. HLA class II molecular typing by PCR-SSO |
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278 | (4) |
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90. HLA-DRB DQB1 molecular typing by PCR-SSP RFLP |
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282 | (2) |
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91. HLA-DPB1 molecular typing by PCR-SSP RFLP |
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284 | (2) |
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92. Detection of complement levels by hemolytic assay |
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286 | (4) |
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93. Hemolytic assay of a single component of complement |
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290 | (4) |
PART FOUR APPENDICES |
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294 | (31) |
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A1. The most common buffers |
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294 | (6) |
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A2. Proteases and protease inhibitors |
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300 | (2) |
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A3. Amino acids and genetic code |
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302 | (2) |
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A4. Protein quantification |
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304 | (4) |
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A5. SDS-PAGE gel staining and drying |
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308 | (4) |
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A6. Substrates for immunoenzymatic reactions |
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312 | (4) |
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A7. Culture media, sera, and additives |
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316 | (4) |
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A8. Sites of interest on the World Wide Web |
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320 | (5) |
Index |
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325 | |