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 packSupplies a direct current between electrode in the electrophoresis unit.

_electrophoresis unitAvailable for running either vertical or horizontal

Gel system.

vertical

horizontal

Two glass plates that are clamped together but held apart by plastic .spacer

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.

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 tertiaryStructure

Principle:

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 pouredOn 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 separateThe 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 .

AcrylamideConcentration)%(

Protein fractionationMr×10ˉ

560-350

1015-200

1510-100

The relationship between acrylamide gel concentration

and protein fractionation range

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 .