4 Molecular Biology Experiment 209

1 Understanding Molecular Biology Experiments 210
2 Preparation and cultivation of microbial growth medium 212
Experiment 1: Preparation and Sterilization of Microbial Growth Media 212
3 Reverse transcription and PCR reaction 216
Experiment 2: Polymerase Chain Reaction (PCR) 216
Experiment 3: Reverse transcriptase polymerase chain reaction (RT-PCR) 220
Experiment 4 Real-time polymerase chain reaction (real-time PCR) 224
4 Plasmid DNA Analysis Techniques 228
Experiment 5 Isolation of Plasmid DNA 228
Experiment 6: Restriction Enzyme Digestion and Confirmation of Plasmid DNA 232
Experiment 7: Electrophoresis 235
5 Applications of Protein Quantification and Antigen-Antibody Analysis 239
Experiment 8 Protein Quantitative Method 239
Experiment 9: Direct ELISA (enzyme-linked immunosorbent assay, ELISA) 244
Experiment 10: Enzyme-Linked Immunosorbent Assay - Indirect ELISA 248
Experiment 11 Enzyme-Linked Immunosorbent Assay - Sandwich ELISA 252
Experiment 12 Biotin labeling of antibodies 257
Experiment 13: Enzyme-Linked Immunosorbent Assay - Competitive ELISA 261
6 The Significance and Utilization of Molecular Biology Experiments 265

5 Proteomics Experiment 267

1 Understanding Proteomics Experiments 268
2 Confirmation of changes in protein expression in animal cells 271
Experiment 1: Confirming Changes in Protein Expression in Animal Cells (1) - Cell Lysis and Total Protein Extraction 273
Experiment 2: Confirming Changes in Protein Expression in Animal Cells (2) - Protein Quantification and Sampling 279
Experiment 3: Confirming Changes in Protein Expression in Animal Cells (3) - Creating an SDS-PAGE Gel 285
Experiment 4: Confirming Changes in Protein Expression in Animal Cells (4) - SDS-PAGE and Transfer 292
Experiment 5: Confirming Changes in Protein Expression in Animal Cells (5) - Ponshu S Staining and Western Blotting 298
Experiment 6: Confirming Changes in Protein Expression in Animal Cells (6) - Film Development and Analysis of Protein Expression Levels 303
3 Confirmation of protein-protein interactions in animal cells 307
Experiment 7: Screening of Binding Proteins by Immunoprecipitation 308
4 Expression and purification of fusion proteins in animal cells 314
Experiment 8: Confirmation of Purification of Fusion Protein 315
Experiment 9: Confirmation of the elution of the fusion protein 320
5 Identification and Use of Phosphorylation Sites in Animal Cells 323
Experiment 10: Detection of Phosphorylated Proteins Using Anti-phosphorylated Antibodies (1) 325
Experiment 11: Detection of Phosphorylated Proteins Using Anti-Phosphorylated Antibodies (2) 329
Experiment 12 Introduction and Application of Phosphorylation Site Mutants 333
6 The Significance and Utilization of Proteomics Experiments 338

6 Immunology Experiment 339

1 Understanding Immunology Experiments 340
2 Observation of Antigen-Antibody Reactions and Immune Cells in Blood 342
Experiment 1: Blood Type Determination Experiment Using Antigen-Antibody Reactions (ABO Blood Typing) 342
Experiment 2: Analysis of Blood Cells Using Wright-Giemsa Stain (347)
3 Understanding and Experimenting with Allergic Immune Responses 352
Experiment 3: Culture of mast cells (key effector cells in allergy) 352
Experiment 4: IgE-mediated mast cell degranulation (β-hexosaminidase release assay: degranulation of mast cells) 357
4 Experimental study on macrophage activation in inflammatory immune responses 365
Experiment 5 Isolation of bone marrow-derived immune cells
immune cells) 365
Experiment 6 Differentiation of bone marrow-derived macrophages 370
Experiment 7 LPS-induced nitric oxide production from macrophages 375
5 Understanding and Morphological Analysis of Lymphatic Tissue 380
Experiment 8: Analysis of morphological features of lymphoid tissues: thymus, spleen 380
Experiment 9: Analysis of morphological features of lymphoid tissues: lymph nodes 385
6. Isolation and Analysis of Immune Cells Using Flow Cytometry 390
Experiment 10: Isolation of spleen-derived immune cells for flow cytometric analysis (splenocytes isolation for flow cytometric analysis) 390
Experiment 11: Pretreatment of immune cells for flow cytometric analysis 394
Experiment 12: Understanding Flow Cytometers 400
Experiment 13 Analysis of lymphocytes, granulocytes, and monocytes using flow cytometry (405)
Experiment 14: Analysis of helper (CD4+) or cytotoxic (CD8+) T cells using flow cytometry [410]
7 The Significance and Utilization of Immunological Experiments 417
Search 419