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Ch. 9 - Techniques for Studying Cells A summary of the chapter and lecture notes on techniques for studying cells, and what's inside of them. Includes illustrations and graphics from the textbook.

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York University
BIOL 2021
Julie Clark

Biol 2021- March 10 2009 lecture Techniques for studying cells (what’s inside) i. Cell prep: collect tissues , grow cells in culture - In vitro: in glass; in vivo: in life conditions - Primary culture: taken directly from animal  Limited life span  Requires immortalizing (using mutations, virus) to grow forever ii. Cell fractionation - Break open cells - Differential centrifugation: separates by size and density (heavy particles go to the bottom) - Can be done multiple times to get smaller and smaller particles isolated Figure 1ultracentrifuge iii. Protein purification: - Chromatography  Column chromatography: protein solution is passed through a column containing a porous solid matrix; can be:  Ion exchange: separates according to charge; good for charged particles – depends on ionic strength and pH of the solution.  Gel filtration: separates based on size  Affinity chromatography: separate based on molecules ability to bind to particular small molecules or to other macromolecules. iv. Once proteins are purified - Polyaccrylamide gel in electrophoresis  Separates based on size - SDS: detergent that gives negative charge - Small fragments migrate faster and further toward the + charged end; while larger molecules migrate slower and less far - Band fragments can be stained and analysed - Provides information regarding the molecular weight and the subunit composition of molecules. v. Protein-Protein interactions - Identifies all of the other proteins to which the protein of interest is bound - Can be studied using co immino precipitation: uses antibodies to pull proteins out of solution  Antibody against 1 specific protein  Antibody recognizes specific target protein; reagents that bind to the antibody and are coupled to a solid matrix then drag the complex out of solution at the bottom of the test tube. If another protein is associated with the antibody then it too will be precipitated out of the solution.  With co immune precipitation proteins are also pulled out with the specific protein - Can also use affinity chromatography to identify protein-protein interactions vi. Small molecule inhibitors - Uses known inhibitors as probes to identify molecules to which an inhibitor binds - Inhibitor to particular protein is put on cells and we observe what happens to process i.e. Kinesin inhibitor with mitosis Figure 2B. shows normal mitosis C. shows mitosis treated with inhibitor kinesin CHAPTER 10: MEMBRANE STRUCTURE Function of membranes: i) Barrier to water soluble molecules; blocks water and keeps it out of cell ii) Separates inside from outside - Cell from environment - Organelles from cytosol - Maintains ion gradient (which is used for energy conversion and signalling) - Signalling  Plasma membrane receptors Structure; - Two layers (lipid bilayer) - Lipid bilayer and proteins - 50:50 lipid to protein weight ratio (ratio varies according to specific membrane function) Membrane contains three major classes of membrane lipids: A) Lipids - Fatty molecule - All lipids are amphiphilic: hydrophobic and hydrophilic - 3 major types of lipids in membrane: phosphoglyceride - a) phosphatidylethanolamine b) phosphatidylserine c) Lipids phospholipids posphatidylcholine Sphingomyelin Phospholipids: most abundant lipid in membrane - Has polar head group and two hydrophobic hydrocarbon tails - One hydrocarbon tail usually has a kink resulting from a cis double bond. - Length and saturation (double bonds) of hydrocarbon tails determine how tightly lipids are packed together (membrane fluidity) - Main types of phospholipids in animal cell membranes are phosphoglycerides.  Phosphoglycerides: phospholipids that contain 3-carbon glycerol backbone and two long chain fatty acids that are linked through ester bonds to adjacent carbon atoms of the glycerol and the third carbon atom is attached to a phosphate group, which in turn can have several different head groups (ie. Choline)  Phosphoglyceride: glycerol+ 2 fatty acid chains+ head group  Polar head groups include choline, phosphate and glycerol  Another important phospholipid is sphingomyelin  Built from sphingosine rather than glycerol  Sphingosine + one fatty acid + choline head group B) Cholesterol - Huge amounts in membrane - Sterol - Contains a rigid ring structure to which is attached a single polar hydroxyl group and a short non polar hydrocarbon chain - Cholesterol: rigid ring + non polar hydrocarbon tail + polar head group (containing hyd
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