Subcellular Fractionation

Today
Background on technique
Purpose
Why it works
General protocol
Homogenization
2 methods of centrifugation
Analysis of fractions
Experiment

Subcellular Fractionation
Mechanism of protein synthesis
DNA replication and transcription
RNA splicing
Muscle contraction
Microtubule assembly
Vesicular transport in the secretory pathway
Importance of mitochondria and chloroplasts
Subcellular Fractionation
Functional studies of organelles and
macromolecular complexes require
detailed observations and manipulations
which are usually impossible with whole
cells.
Means to help separate complicated
mixtures based on sizes, shapes, density,
viscosity of medium, rotor speed, with help
of gravity.
Important implications
Study single biological processes free from other
interfering reactions in the cell.
Dont have to worry about keeping the cells
intact and alive: cell-free system.
Fractions are usually biologically active and can
be stored for later use in freezer.
Minimal equipment is needed and starting
material is usually easily obtained.
Most protocols have been worked out, but may
some minor adjustments.
Determination of Protocol
Know your goal.
Do you need enzyme activity?
Are you looking for composition or morphology?
Are you isolating a specific organellar protein?
What is your material?
Tissue, cultured cells, yeast, bacteria can be used.
Use the gentlest homogenization procedure for your
starting material.

Sedimentation of a molecule is
influenced by:

Properties of the molecules (size, shape,
density).
Properties of the solvent, or the gradient
material (density, viscosity, temperature).
Interactions between the solute molecules
and the solvent gradient material.
Multi-Step Procedures
Homogenization
Differential Centrifugation
Further separation and purification by
density gradient centrifugation if needed
Fraction collection and analysis

Equipment
Homogenizer of some form
Centrifuge: low and high speed
Appropriate rotors: fixed angle or swinging
bucket
Spectrophotometer (for measuring
[protein])
Method to check and evaluate different
fractions (Western blot or enzymatic
activity for organelle markers)

Homogenization Methods
Osmotic shock: hypotonic buffer makes cells
swell and burst.
Sonication: sound waves to lyse cells.
Mechanical shearing or grinding: blenders,
Dounce homogenizers, Polytron.
Buffers usually isotonic or hypotonic, some sucrose,
buffered with Tris-HCl, pH 7.4 with protease inhibitors
added and EDTA to chelate Mg
2+
or Ca
2+.
Ideal to do on ice and at 4
o
C.
GOAL: Break up plasma membrane to release
cellular organelles intact.
Centrifugation Steps
Differential: usually the first step in the
approach which crudely separates
particles on the basis of size: our
approach because of time.

Density gradient: separates particles
based on density and size to yield pure
organelle fractions.
Centrifugal Force
Particles in suspension can be separated
by sedimentation velocity (differential) or
sedimentation equilibrium (density
gradient).
Sedimentation depends on molecular size,
shape, density, but also frictional force and
diffusion force.
Animal Cell
Rough ER Smooth ER
Centrosome
CYTOSKELETON
Microfilaments
Microtubules
Microvilli
Peroxisome
Lysosome
Golgi apparatus
Ribosomes
In animal cells but not plant cells:
Lysosomes
Centrioles
Flagella (in some plant sperm)
Nucleolus
Chromatin
NUCLEUS
Flagelium
Intermediate filaments
ENDOPLASMIC RETICULUM (ER)
Mitochondrion
Nuclear envelope
Plasma membrane
Campbell and Reece, 2005
Differential Centrifugation
Density of solvent is uniform.
Density of solvent << Density of
particles.
Viscosity of the solvent is low.
Consequence:
Rate of particle sedimentation
depends mainly on its size and the
applied g-force.
Differential Centrifugation
Differential Centrifugation
Low speed: 1,000 times gravity (10 minutes)
Pellet contains whole cells, nuclei, cytoskeleton
Supernatant: further centrifugation
Medium Speed: 20,000 times gravity (20 minutes)
Pellet contains mitochondria, lysosomes and
peroxisomes
Supernatant: further centrifugation
High Speed: 80,000 times gravity (60 minutes)
Pellet contains microsomes and small vesicles.
Supernatant: further centrifugation
Very High Speed: 150,000 times gravity (3 hours)
Pellet contains viruses, ribosomes, large
macromolecules

Size of major cell
subsructures from liver
tissue
Nucleus 4-12 m
Large plasma membrane 3-20 m
fragments
Mitochondria 0.4-2.5 m
Lysosomes/peroxisomes 0.4-0.8 m
Microsomal vesicles 0.05-0.3m
Our approach
Low speed: 1,000 times gravity (10 minutes)
Pellet (P1) contains whole cells, nuclei,
cytoskeleton: discard
Supernatant (S1): further centrifugation
Medium Speed: 20,000 times gravity (20
minutes)
Pellet (P2) contains mitochondria,
lysosomes and peroxisomes
Supernatant (S2): cytosolic proteins and
small organelles

S2 fraction from Rat Tissue
Soluble cytoplasmic fraction composed of
smallest organelles, small plasma
membrane fragments, and cytoplasmic
proteins.
Crude P2 Pellets from Rat Tissue

lysosomes (0.4-0.8m), peroxisomes (0.4-
0.8 m, Golgi (0.05-0.5 m), rough
endoplasmic reticulum (0.05-0.35 m).
Density Centrifugation
Two Types:
Rate-Zonal
Isopycnic
Different types of media to make gradient
to be used to obtain desired end: Ficoll,
Percoll, Sucrose, CsCl

Rate- Zonal Centrifugation

Makes use of a continuous density gradient of
solvent such as sucrose.
Density of sucrose increases towards the bottom
of the tube.
Sample is layered on the top.
Molecules form discrete bands (zones) after
centrifugation.
Separation is based on size of the molecules.
Swinging bucket rotors
Isopycnic Separation

Based on the density of the molecules, not
size.
The sample molecule can be layered on
density material.
Molecules move to the position where their
density is same as the gradient material
Swinging bucket or fixed angle rotor
How does a gradient
separate different particles?
Least dense
Most dense
Density Gradient
A particle will sediment through a
solution if particle density > solution
density
If particle density < solution density,
particle will float through solution
When particle density = solution
density the particle stop sedimenting or
floating

1

5

2

3

4

1 2 3 4 5
Fixed Angle Swinging Bucket
Collecting Fractions
KEEP SAMPLES PURE!!!!!
Try to avoid cross contamination of
supernatant and pellet fractions.
Analysis of Fractions
May need to identify and quantify fractions for
further use in other protocols and separations.
Light microscopy
Determine protein concentration by various methods
(spectrophotometer, BCA assay, Bradford assay)
Determine activity of enzyme of interest if any in
relation to protein concentration
Assay for protein marker by PAGE with western blot
using antibodies
Assay for the presence of marker enzymes in
appropriate fractions and look for any cross-
contamination between fractions

Marker Enzyme Assays for
Organelles
The term 'marker' is employed when an
antigen is known to be expressed in a specific
cell, tissue or sub-cellular location.
ER PDI (Protein Disulfide Isomerase)
Golgi Galactosyl transferase
Lysosomes Beta-galactosidase
Mitochondria Succinate dehydrogenase
Peroxisomes ????? You tell me.
PM ????? You tell me.
Lysosomes ????? You tell me.
Tissue
Homogenization
Homogenate
1000 g
(1000 times the
force of gravity)
10 min
Differential centrifugation
After each run, supernatant poured into next tube
20,000 g
20 min
Pellet rich in
nuclei and
cellular debris
Pellet rich in
mitochondria
(and chloro-
plasts if cells
are from a
plant)
Pellet rich in
microsomes (pieces
of plasma membranes
and cells internal
membranes)
Pellet rich in
ribosomes
150,000 g
3 hr
80,000 g
60 min
Modified from Campbell and Reece, 2005
Our samples
Thank you Dr. Rhoads!!
Either Long Evans (LE) Rat Liver, Kidney,
Heart Tissue.
Exposed to different environmental
conditions (alcohol, caffeine,
amphetamine) over 2-3 week period.
Some tissue is taken from rats that were
going through amphetamine withdrawl.

Our Future Goals
Determine protein concentration of H, S2 and P2 fractions by Bradford Assay
Use these fractions to isolate
Glutathione S-transferase
(GST) isoforms (antioxidant proteins))
by GST pull-downs to
determine localization of enzyme
and effect of diet on expression
of GST
Use these fractions to assay
Catalase activity or GST activity
(antioxidant protein) to determine
localization of catalase or GST in
cell.
H, Supernatants (S2) and Pellets (P2)

Our Future Goals
Determine protein concentration by Bradford Assay
Homogenate

Use homogenate to determine
presence of mRNA for GST to see
if there is a correlation to
protein amount observed by
SDS-PAGE and if there is a
difference caused by diet.
Our Ultimate Goal
Determine the effects of these different
environmental conditions on
expression levels of GST protein (and
mRNA levels) in tissues and enzyme
activity of either GST and/or catalase.