RECENT TRENDS IN MOLECULAR BIOLOGY

TECHNIQUES
Course No.: MBFS 514
Course Rating: 3 Credits

DR. MUHAMMAD FAROOQ SABAR

Associate Professor
Center for applied molecular biology university of the
Punjab Lahore.
•
Course
1. Principal and Applications:
Contents:
General procedures and instrumentation in centrifugation, electrophoresis,
chromatography, Ion exchange chromatography, molecular sieve chromatography,
affinity chromatography, paper chromatography, thin layer chromatography,
ultrafiltration, Ultracentrifugation, spectrophotometry, spectroscopy, microscopy,
general techniques in microbiology, PCR, Real-Time PCR.
2. Isolations:
Protein/peptide separation techniques, Cell disintegration, and extraction techniques,
and separation of proteins by fractionation (ammonium sulfate, organic solvents).
3. Analysis:
MALDI-TOF, HPLC, FPLC, FT-IR spectroscopy for the identification of biological
stains for forensic purposes, applications of quantitative enzyme assay, enzyme
activity, and specific activity determination Nucleic acid & protein blotting
techniques. DNA Sequencing
 What is Centrifuge ?
A device for centrifugation -
Separation
Separating particles
Separation of particles depends
on
Centrifuge works on principle of
centrifugation.
Centrifugation occur by
centrifugal force.
Centrifugal force sediment
heterogeneous mixture.
• Size
• Shape
• Density
• Viscosity
• Speed
• Radius of rotation Object moving in circle at any velocity
is forced to outward direction.
Outward force (F = RCF=g-Force)
depends on
•Speed of centrifuge machine
•Radius (rotor radius) = r
•Molecular weight of particles
 Principle
Centrifugal action creates an induced gravitational force in an outward direction
relative to the axis of rotation and this drives the particles or precipitate towards the
bottom of the tube.
 Major Topics

• Common centrifuge rotors

• Calculation of g-force
• Differential centrifugation
• Density gradient centrifugation
Types of Centrifuge Machines
Based On Rotor Speed
 Types of Centrifuge Machines
Based on Rotor Angles

Swinging Bucket Rotor

Vertical Rotor

Fixed Angle Rotor

 Centrifuge rotors
axis of rotation
Swinging-bucket

At rest Spinning g
g

Fixed-angle
 Geometry of rotors
rmax rav rmin axis of rotation
a
rmin rmin
rav rav
rmax rmax
b c

Sedimentation path length

 Calculation of RCF and Q
 Q 2
RCF = 11.18 x r  
 1000 
RCF
Q = 299
r
RCF = Relative Centrifugal Force (g-force)
Q = rpm; r = radius in cm
Measuring the Radius of Centrifuge Rotor
Measuring the radius
of the centrifuge.
The distance
measured from the
rotor axis to the tip of
the liquid inside the
tubes at the greatest
horizontal distance
from the rotor axis is
the rotating radius.
The measurement
length must be in
centimeters.
Measuring the Radius of Centrifuge Rotor

The distance
measured from the
center of the rotor
to the bucket edge
is the rotating
radius in
centimeter.
Measuring the Radius of Centrifuge Rotor

The radius is
measured
from the axis
of rotation to
the inner
bottom of the
swing-out
cup.
Graphical Representation of
RCF-RPM
 Velocity of sedimentation of a particle

d (  p − l )
2

v=
18
v = velocity of sedimentation
p = density of particle
g = centrifugal force
g
d = diameter of particle
l = density of liquid
 = viscosity of liquid
 Differential centrifugation
• Density of liquid is uniform
• Density of liquid << Density of particles
• Viscosity of the liquid is low

Rate of particle sedimentation depends

mainly on its size and the applied g-force.
 Size of major cell organelles
• Nucleus 4-12 m
• Plasma membrane sheets 3-20 m
• Golgi tubules 1-2 m
• Mitochondria 0.4-2.5 m
• Lysosomes/peroxisomes 0.4-0.8 m
• Microsomal vesicles 0.05-0.3m
Differential centrifugation of a
tissue homogenate (I)
Decant
1000g/10 min supernatant

etc. 3000g/10 min

Differential centrifugation of a
tissue homogenate (II)
1. Homogenate – 1000g for 10 min
2. Supernatant from 1 – 3000g for 10 min
3. Supernatant from 2 – 15,000g for 15 min
4. Supernatant from 3 – 100,000g for 45 min
• Pellet 1 – nuclear
• Pellet 2 – “heavy” mitochondrial
• Pellet 3 – “light” mitochondrial
• Pellet 4 – microsomal
Differential centrifugation (III)
• Poor resolution and recovery because of:
• Particle size heterogeneity
• Particles starting out at rmin have furthest to
travel but initially experience lowest RCF
• Smaller particles close to rmax have only a
short distance to travel and experience the
highest RCF
Differential centrifugation (IV)

Swinging-bucket rotor:
Long sedimentation path length
gmax >>> gmin

Fixed-angle rotor:
Shorter sedimentation path
length
gmax > gmin
 Differential centrifugation (V)
• Rate of sedimentation can be modulated by
particle density
• Nuclei have an unusually rapid
sedimentation rate because of their size
AND high density
• Golgi tubules do not sediment at 3000g, in
spite of their size: they have an unusually
low sedimentation rate because of their very
low density: (p - l) becomes rate limiting.
 Typical differential centrifugation parameters for a biological sample (path length of centrifugation ≈1–5 cm)

Sample input G force Time Instrument needed Pellet contents Supernatant

contents
Unlysed 100 x g 5 min Benchtop fixed- Intact (eukaryotic) Varies depending on
(eukaryotic) cells angle centrifuge, or cells, macroscopic sample
swinging bucket debris
centrifuge

Gently lysed cells 600 x g 10 min Benchtop fixed- Nuclei Cytosol, non-nuclei
(e.g. dounce angle centrifuge, or organelles
homogenizer) swinging bucket
centrifuge

Supernatant of 15,000 x g 20 min Benchtop fixed- Mitochondria, Cytosol, microsome

previous row angle centrifuge chloroplasts, s (known as post
lysosomes, mitochondrial
peroxisomes supernatant)

Supernatant of 50,000 x g - 60 min High speed fixed- Plasma membrane, Cytosol, ribosomal
previous row 100,000 x g angle centrifuge, or microsomal subunits, small
vacuum fraction, large polyribosomes,
ultracentrifuge polyribosomes enzyme complexes

Supernatant of 50,000 x g - 120 min Vacuum Ribosomal subunits, Cytosol

previous row 100,000 x g ultracentrifuge small poly
ribosomes, some
soluble enzyme
complexes
 Density gradient centrifugation
Density Barrier Discontinuous Continuous
How does a gradient separate
different particles?
Least dense

Most dense
Predictions from equation (I)

d (  p − l )
2

v= g
18
When p > l : v is +ve
When p = l : v is 0
Predictions from equation (II)

d (  p − l )
2

v= g
18
When p < l : v is -ve
 Summary

• 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

2 Buoyant density
banding
3
Equilibrium
density banding
4
Isopycnic
5 banding
 3 Formats for separation of particles according
to their density

When density of particle < density of liquid V is -ve

Resolution of density gradients

Density Barrier Discontinuous Continuous

I II
Separation of particles according to size

p >> l : v is +ve
for all particles
throughout the
gradient