Transcript Electrophoretic_techniques2003

Electrophoretic techniques
Introduction:
_The term electrophoresis describe the
migration of a charged particle under the
influence of an electric field.
Many important biological molecules such as
amino acid, peptides, proteins. Nucleotides
and nucleic acids, possess ionisable groups
and therefore,
At any given PH, exist in a solution as electrically
charged species either as a cation (+) or
anion(-).
Under the influence of an electric field these
charged particles will migrate either to
cathode or anode, depending on the nature of
their net charged
The equipment required for electrophoresis
consist basically of two items,
_power pack
Supplies a direct current between electrode in
the electrophoresis unit.
_electrophoresis unit
Available for running either vertical or
horizontal
Gel system.
vertical
horizontal
Two glass plates that are
clamped together but held
spacer.apart by plastic
A plastic comb is placed in
gel solution
And removed after
ploymerisation to provide
loading wells for up to 10
samples.
When apparatus is
assembled, the lower
electrophoresis tank buffer
surrounds the gel plates
and affords some cooling of
gel plates.
kinds of gels for gel electrophoresis
The gel is a polymer whose composition and •
porosity is chosen based on the specific
weight and composition of the target to be
analyzed (the gel is a solid, yet porous matrix) •
Two kinds of gels are commonly used: •
1. agarose •
2. polyacrylamide. •
SDS-polyacrylamide gel
electrophoresis
SDS-PAGE is the most widely used method for
analysing protein mixtures qualitatively.
the method is based on the separation of protein
according to size.
Because of that,
It’s particularly useful for monitoring protein
purification and used to determine the relative
molecular mass of protein.
Principle:
SDS (CH3-(CH2)10-CH2OSO3-Na+) is an anionic
detergent.
Samples to be run on SDS-PAGE are firstly boiled for
5 min in sample buffer containing
β-mercaptoethanol and SDS.
The mercaptoethanol reduces any disulphide
Bridge present that are holding the protein tertiary
Structure
and SDS bind strongly to, and denatures the
protein.
Each protein in the mixtrue is therefore fully
denatured by this treatment and opens up
into a rod-shaped structure with a series of
-vely charged SDS molecules along polypeptide
chain.
The sample buffer contain ionisable tracking
dye,usually bromophenol blue, that allows the
electrophoretic run to be monitored.
sucrose or glycerol that give the sample solution
density thus, allowing the sample to settle
easily through the electrophoresis buffer to
the bottom when injected into loading gel.
Once the sample are loaded, a current is passed
through the gel.
The sample to be separated are not infact
loaded directly into a main separating gel has
been poured between the glass plates and
allowed to set a shorter stacking gel is poured
On top of separating gel and it is into this gel
that the wells are formed and protein loaded.
The purpose of this stacking gel is to
concentrate the protein sample into a sharp
band before it enters the main separating gel.
The stacking gel has a very large pore size which
allow the proteins to move freely and
concentrate or stack under the effect of
electric field.
When current is switched on, all the ionic
species have to migrate under influence of
electric field.
as they pass through the separating gel, the
protein separate
The smaller protein move more easily whereas
Large proteins are successively retarted by
frictional resistance due to sieving effect of the
gel.
The bromoethanol blue dye is totally un retarted
and therefore indicates the electrophoresis
front.
When the dye reaches the bottom of the gel,
The current is turned off,and the gel is removed
from between the glass plates and shaken in
an appropriate stain solution (usually
coomassie brilliant blue )
And then washed in destain solution to remove
unbound background dye from the gel, leaving
proteins visible as blue band stained on a clear
back ground.
The choice of gel to be used is depend on the
size of protein being studied.
The relationship between acrylamide gel concentration
and protein fractionation range
Acrylamide
Concentration(%)
Protein fractionation
Mr×10ˉ
5
60-350
10
15-200
15
10-100
The Mr of a protein can be determined by
comparing it’s mobility with those of a
number of standard protein of known Mr
that are run on the same gel.
By blotting a graph og distance moved against
log Mr for each of standard protein.
Acalibration curve can be constructed.
The distance moved by the protein of unkown
Mr is then measured.
And then it’s log Mr and hence Mr can be
determined from the calibration curve.
SDS-gel electrophoresis is often used after
Each step of purification protocol to assess
the purity or otherwise of the sample.
A pure protein should give a single band on an
SDS-poly acrylamide gel, unless the molecule
is made of two unequal subunits.
In the latter case two bands, corresponding to
the two subunits will be seen
Since only submicrogram amount of protein are
needed for the gel.