part i.3 methods in molecular cell biology 304515 molecular biology of the cell teacher: montarop...

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