This document summarizes agarose gel electrophoresis and ultracentrifugation techniques. It discusses how agarose gel is used to separate biomolecules like proteins and nucleic acids based on their size and charge by running an electric current through the gel. Ultracentrifugation uses high centrifugal forces to separate particles based on properties like density, shape and size. There are two main types - analytical ultracentrifugation which studies molecular interactions, and preparative ultracentrifugation which isolates and purifies organelles and particles through techniques like density gradient centrifugation.

1. AGAROSE GEL AND ULTRA
CENRTIFUGATION
Presented by :- Patel Rinku D .
CBO :- 406
M.Sc Sem :- 2
Department of life sciences ,
H.N.G.U., Patan

2. Content
• Agarose gel electro – phoresis and electro-focusing
• Ultra centrifugation :
 Principles and types

3. Agarose Gel electrophoresis and electro-
focusing
• Agarose is a linear polysaccharide made up pf the basic repeat
unit agarobiose ,which comprises alternating units of galactose
and 3,6 –anhydrogalactose .
• Agarose is one of the components of agar that is a mixture of
polysaccharides isolated from certain seaweeds .
• Agarose is usually used at concentrations of between 1% and 3%
agarose gels are formed by suspending dry agarose in aqueous
buffer ,then boiling the mixture the until a clear solution forms .
• This is poured and allowed to cool to room temperature to form a
rigid cells .

4. Agarose gel electrophoresis

5. • The gelling properties are inter and intramolecular hydrogen bonding
within and between the long agarose chains .
• The pore size in the gel is controlled by the initial concentration of
agarose ; large pore sizes are formed from low concentrations and
smaller pore sizes are formed from the higher concentration .
• Agarose gels are used for the electrophoresis of both proteins and
nucleic acids for proteins , the pore sizes of a 1% agarose gel are large
relative to the size of proteins .
• Agarose gels are therefore used in techniques such as
immunoelectrophoresis or flat bed isoelectric focusing, where the
proteins are required to move unhindered in the gel matrix according
to their native charge .

6. • Such large pore gels are also used to separate much larger
molecules such as DNA or RNA .
• A further advantage of using agarose is the availability of low
melting temperature agarose ( 62-65·C).
• The gel rod system sometimes used for acrylamide gels is not
used .
• Horizontal slab gels are invariably used for isoelectric focusing
or immunoelectrophoresis in agarose .
• Horizontal gels are also used routinely for DNA and RNA gels .

7. Ultra-centrifugation
 Introduction
 Centrifuge :-
• A centrifuge is a device for separating particles from a solution
according to their size , shape, density, viscosity of the medium
and rotor speed .
• Svedberg coined the term “ ultracentrifuge” .He was colloid
chemist .
• The first commercial ultracentrifuge was produced in 1940 by
SPINCO.

8. Principle
• Sedimentation of solute particles makes heavier particles sink
and lighter particles to float in the solvent or medium .
• The solution is subjected to high angular velocity measured in
rpm ,the effect of gravity and thereby the rate of
sedimentation greatly increases due to application of an
external force called centrifugal force .
• Centrifugal force is defined as
F = mw2r
Where , F = intensity of centrifugal force
m = effective mass of sedimenting particles
w = angular velocity of rotation
r = distance of migrating particles from central
axis of rotation

11. Types
1 . Analytical ultracentrifugation :-
• The aim of analytical ultracentrifugation is use to study
molecular interactions between macromolecules or to analyse
the properties of sedimenting particles such as their apparent
molecular weight .
2 . Preparative ultracentrifugation :-
• The aim of preparative ultracentrifugation to isolate and
purify specific particles such as subcellular organells .

13. Analyitical ultracentrifugation
• Two kinds of experiments are commonly performed On these
instruments :-
i. Sedimentation velocity experiments :-
 Aim of SVEs to interpret the entire time - course of sedimentation ,
and report on the shape and molar mass of the dissolved
macromolecules , as well as their size distribution .
ii. Sedimentation equilibrium experiments :-
 SEEs are Concerned only with the final steady – state of the
experiment , where sedimentation is balanced by diffusion
opposing the Concentration gradients , resulting in a time –
independent Concentration profile .

14. Preparative ultracentrifugation
•It is isolate specific particles which can be reused .
1 . Differential ultracentrifugation :-
 Differential centrifugation is a common procedure in microbiology and
cytology used to separate certain organelles from whole cells for further
analysis of specific parts of cells .
 In this process , a tissue sample is first homogenised to break the cell
membrane and mix up the cellular contents .
 Mixture of homogenate particles can be separated by centrifugation on
the basis of their densities or size .
 Crude tissue homogenates containing organelles , membrane vesicles and
other structural fragments are divided into different fractions by the
stepwise increase of the applied centrifugal field .

16. 2 . Density gradient
ultracentrifugation
To further separate biological particles
of similar size but differing density ,
ultracentrifugation with per-formed or
self-establishing density gradients is the
method of choice . Both are separation
or equilibrium methods can be used .
Based on density difference . There are
two types of density gradients
ultracentrifugations under preparative
ultracentrifugation such as :-
1 . ZONAL or RATE 2 . ISOPYCNIC

17. I. ZONAL or RATE Centrifugation :
 Mixture to be separated is layered on top of a gradient (
increasing Concentration down the tube ) .
 Provides gravitational stability as different species .
 Move down tube at different rates .
II. ISOPYCNIC Centrifugation :-
 Isopycnic means “of the same density . “
 Molecules separated on equilibrium position .
 Each molecule floats or sinks to position where density .

18. • References :-
 Principles and Techniques of Biochemistry and Molecular
Biology By ,
Keith Wilson and
John Walker
https://www.ncbi.nlm.nih.gov

19. Thank You