BIOCHEMICAL TECHNIQUES
Introduction
-
These
methods are used by biologists, biotechnologists, genetic engineers,
pharmacologists and others for the separation of one kind of biomolecules from
other.
-
Specific
techniques are used for separation of specific molecules.
-
These
techniques exploit the physical, chemical and electrical properties of
molecules for their separation.
-
These include a large number of techniques
like centrifugation, chromatography, gel penetration techniques,
electrophoresis, spectrometry and many more.
CENTRIFUGATION
INTRODUCTION
-
This
method of separation is based on sedimentation rate.
-
Sedimentation
rate depends upon the size and shape of the molecule and the viscosity of the
solution.
-
Higher
the molecular weight greater the rate of sedimentation.
-
In
centrifugation we rotate a suspension at a very high speed so that the
components settle down quickly.
-
When
a suspension is centrifuged the components of the suspension of the suspension
are separated into pallets and supernatant.
-
Every
suspension has to be centrifuged at a specific speed for a specific time for
its separation.
Types of
Centrifugation Based On Density
-
RATE ZONAL
o
The separation of molecules is based on the
sedimentation coefficient of the molecule.
-
ISOPYCNIC
o
Separation
on basis of buoyant density.
-
DENSITY BARRIER
Types of
Centrifugation Based On Speed (RPM)
-
Low speed centrifugation
o
RPM
is between 3000-6000.
o
RCF
is 6000g.
o
Large
organelles like nucleus and chloroplast are separated.
-
Micro speed centrifugation
o
RPM
is 12000.
o
RCF
is 10000g
-
High speed centrifugation
o
RPM
is 25000.
o
RCF
is 60000g.
o
Used
to separate mitochondria and lysosomes.
-
Ultracentrifugation
o
RPM
is 30000.
o
RCF
id 60000g.
o
Used
to separate ribosomes, vesicles and other biological macromolecules.
GELPENETRATION
INTRODUCTION
-
This
method uses gel beads that are made of inert material and have pores in them.
-
This
method is used when molecules are to be separated based on their size.
-
The
particles with larger size run down fast as they cannot get into pores of the
beads, since smaller particles get into the pores of the beads they take longer
time to finish the run.
Characteristics
of beads
-
Chemically
inert.
-
Uniform
pore and particle size.
-
High
mechanical rigidity.
-
Contains
no ionic groups.
Advantages
of gel penetration
-
Gentleness.
-
Maximum
recovery of sample.
ELECTROPHORESIS
INTRODUCTION
- This is a technique that is used to separate charged molecules based on their mobility in electric field.
- This technique is widely used in the separation of DNA.
- Mobility depends upon the net charge of the molecule, size of the molecule, shape of the molecule and strength of the magnetic field.
WORKING
- This technique uses a medium called Agarose gel.
- The DNA fragments are digested with the help of restriction enzymes and placed in the wells made in the gel setup.
- An electric field is applied across the gel.
- Since the DNA molecule is negatively charged it moves towards the positive electrode.
- The smallest strand of DNA covers the maximum distance in a given period of time than the larger strands.
- The DNA strands can be stained with Ethidium bromide and visualized.
LAMBERT-BEER LAW
When a monochromatic light of intensity I0 passes through a medium there is a change in its intensity depending upon the concentration ‘C’ of the medium and the path length ‘L’ of the sample.
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