Transcript Analytical Approaches in Cell Biology

Analytical Approaches in Cell
Biology
BSCI 420
Sept 12, 2002
“Make everything as simple as possible, but not simpler”
-Albert Einstein
1. Separation of cell components
2. Cell culture, fusion and hybridomas
1. Separatory techniques:
Cells must first be disrupted by homogenization,
osmotic rupture, detergent lysis, or ultrasonic disruption.
A. Centrifugation is useful for separation on the basis of mass
or density.
[A8-7] preparative ultracentrifuge
1) Differential acceleration:
Repeated centrifugation at progressively higher speeds, e.g.
low 1000 X g
med 20,000 X g
high 80,000 X g
very high 150,000 X g
Preparative ultracentrifuge
Differential acceleration
Velocity sedimentation
Equilibrium densitygradient centrifugation
To sep. organelles or
molecules of similar
mass but diff. Densities.
E.g., lysosomes & mitoch.
Or labeled DNA from inlabeled.
2. Chromatography
A) Ion exchange - Charged resin on beads, e.g.
DEAE dextran (+) 0r phosphocellulose (-) charged.
Oppositely charged molecules bind, released by salt solution.
B) Gel filtration - Retards smaller molecules that enter
pores in gel beads, so larger molecules come thru 1st.
C) Affinity chromatography - Specific ligand or antibody
bound to beads. Elute with buffer that dissociates ligand
from protein, e.g. low pH, hi salt
3. Electrophoresis
Separates based on
migration rate in electrical
field.
Most common: SDS-PAGE
Uniformly neg
charged so migrate on the
basis of size
Isoelectric focusing;
Run in solution of ampholines that maintain a pH gradient
2D gel electophoresis: IEF, then SDS-PAGE
2. Cell culture, Fusion , & Hybridomas
3 types of cell cultures:
a) Primary cell cultures- Directly from an organism
b) Normal cell lines - Cells that have been subcultured
and adapted to cell culture (mortal-age and die)
c) Transformed cell lines - Virus, mutated, or derived
from a tumor. (immortal)
All 3 can still retain some different cell f(x)s.
(A) Fibroblasts
(B) Myoblasts and myotubes
(C) Oligodendrocytes
(D) Tobacco cells
Fusion, heterocaryons, and and hybrid cells
Production of
hybridomas and
monoclonal antibodies
Select in HAT med ->
Pos clones -> MCABs