BIOL 2021 Lecture Notes - Affinity Chromatography, Column Chromatography, Lipid Bilayer

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Published on 16 Oct 2011
School
York University
Department
Biology
Course
BIOL 2021
Professor
Biol 2021- March 10th 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
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- 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
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Document Summary

Cell prep: collect tissues , grow cells in culture. In vitro: in glass; in vivo: in life conditions. Requires immortalizing (using mutations, virus) to grow forever. 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. 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. Affinity chromatography: separate based on molecules ability to bind to particular small molecules or to other macromolecules. 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. Identifies all of the other proteins to which the protein of interest is bound.