Part I.3Methods in Molecular Cell Biology
304515 Molecular Biology of the Cell
Teacher: Montarop Yamabhaihttp://www.sut.ac.th/AgriTech/Biotech/mywebpage.html
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
Fractionation of Cells
• Organelles and macromolecules can be separated by ultracentrifugation
• There are 2 types of sedimentations– Velocity sedimentation– Equilibrium sedimentation
DNA Engineeting
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
Determination of size and subunit of a protein by
SDS polyacrylamide-Gel Electrophoresis
Determination of 3D Structure of the Proteins
The study of protein-protein interacton
• Affinity Chromatography and Immunoprecipitation
• Yeast Two-Hybrid System
• Phage Display Method
Studying gene expression and function
• Genetic Screen
• Microarrays
• Targeted mutations
Visualizing Cells
Fluorescence Microscopy
Electron Microscopy
• Transmission electron microscopy TEM
• Scanning electron microscopy SEM
• Electron microscope tomography
Visualize molecules in living cells
• Visualize ion with light-emiting indicator
• Visualize GFP-tagged proteins
• Trace molecules in cells by pulse-chase experiment
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