1. Part I.3 Methods in Molecular Cell Biology 304515 Molecular Biology of the Cell Teacher: Montarop Yamabhai http://www.sut.ac.th/AgriTech/Biotech/mywebpage.html

2. Manipulating cells in the culture Isolating cells –Disrupt extracellular matrix with proteolytic enzymes or EDTA –Fluorescence-activated cell sorter Growing cells in culture dishes –Primary cell culture Prepare directly from tissues of an organism –Cell line Immortalized, can grow indefinitely

6. Fractionation of Cells Organelles and macromolecules can be separated by ultracentrifugation There are 2 types of sedimentations –Velocity sedimentation –Equilibrium sedimentation

11. DNA Engineeting

41. Analyzing Protein Structure and Function Chromatography / HPLC / FPLC –Ion-exchange/hydrophobic chromatography –Gel-filtration chromatography –Affinity chromatography Gel Electrophoresis Western Blotting Mass spectrometry X-ray crystallography/NMR spectroscopy Protein-protein interaction Protein-DNA interaction

47. Determination of size and subunit of a protein by SDS polyacrylamide-Gel Electrophoresis

56. Determination of 3D Structure of the Proteins

60. The study of protein-protein interacton Affinity Chromatography and Immunoprecipitation Yeast Two-Hybrid System Phage Display Method

66. Studying gene expression and function Genetic Screen Microarrays Targeted mutations

77. Visualizing Cells

84. Fluorescence Microscopy

92. Electron Microscopy Transmission electron microscopy TEM Scanning electron microscopy SEM Electron microscope tomography

97. Visualize molecules in living cells Visualize ion with light-emiting indicator Visualize GFP-tagged proteins Trace molecules in cells by pulse-chase experiment