topic 1B_rivier_252 - MCB 252 Topic 1b Techniques and...

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MCB 252 Topic 1b Techniques and Technology Prof. David Rivier For questions on course material and science, see me in person, do not email me!!!
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Reading for this lecture: Protein Purification pp. 96-98 X-ray Crystalography pp. 103-4 Immuno-flourescent Microscopy section 9.2 Antibodies pp. 400-401 Transfection and Transformation pp. 196-7 Transgenic Animals Figure 5-43 GFP pp. 197-8
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Today Technology- Protein purification Antibodies Immunoflorescence GFP Transformation, Transfection, Transgenics X-ray crystalography Actin cytoskeleton
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How do we identify and study the proteins invovled in any particular process? How do we know which proteins are produced in which cell types? How do we know where any given protein is located within a cell?
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Strategies to study the proteins that are invovled in a particular process: Biochemistry: Purify the proteins invovled and study their behavior in vitro . Recapitulate the process in a test tube. Genetics: Isolate a mutant that is defective in the process. Cell Biology: Watch the process in a microscope. Follow the dymanics of particular proteins in a living cell. Does inhibiting the function of a specific protein in a living cell block the process you are interested in? None of these fields are independent: Cell biologists do biochemistry, Cell biologists use genetics, Geneticists use cell biology….
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Basic Techniques for Studying Biology at the Cell and Molecular Level “Biochemistry” Protein How do we purify a protein of interest?
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Fraction A Fraction B Fraction C Distinct Subsets of Proteins Fractionation - Separate by Physical & Chemical properties Cell Extract 1000’s of proteins
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Standard Column Chromatography Protein Elution via [Salt] in Buffer
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How do you know which fraction(s) contain the protein you are interested in?
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