Horizontal gel electrophoresis

Electrophoresis

The Separation of macromolecules under the influence of a uniform electric field through a matrix which is porous in nature is to be termed as ELECTROPHORESIS

Electrophoresis

Zone Electroph

oresisPaper

electrophoresis

Gel electrophoresis

Isoelectric

foucusing

Immunoelectropho

resis

Different types of electrophoresis

• Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

Gel Electrophoresis

• Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins.

• Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA.

Agarose

Detector

•Employs electromotive force to move molecules through a porous gel•Separates molecules from each other on the basis of size and/or charge and/or shape •Basis of separation depends on how the sample and gel are prepared

Gel electrophoresis

Horizontal gel electrophoresis

Vertical gel electrophoresis

Important types of gel electrophoresis

Porous Material

Proteins Entering Porous

Material

Smallest Move Fastest

•Also called Agarose gel electrophoresis•In this gel electrophoresis the matrix used is a gel and is made up of agarose•Agarose – a complex sugar chain from red seaweed. It has a large pore size good for separating large molecules quickly.

Horizontal Gel Electrophoresis

Components of an Electrophoresis System

Power supply and chamber, a source of negatively charged particles with a cathode and anode

Buffer, a fluid mixture of water and ions Agarose gel, a porous material that DNA

migrates throughGel casting materialsDNA ladder, mixture of DNA fragments of

known lengthsLoading dye, contains a dense material and

allows visualization of DNA migrationDNA Stain, allows visualizations of DNA

fragments after electrophoresis

Buffer

Dyes Power Supply

+

-

Agarosegel

Cathode

Anode

Where does the current come from?

A direct current power supplyIons supplied by the bufferThe charge on the macromolecules being

separatedElectrolysis of water

Electrolysis of water4H2O 2H2 + O2 + 2H2O

self-ionization of water throughout the buffer: 4H20 4H+ + 4OH-

At the negative pole4H+ + 4e- 2H2

At the positive pole4OH- O2 + 2H2O + 4e-

What factors affect mobility of linear ds DNA?

Pore size of the gel [agarose] pore size pore size friction mobility

Voltage across the gel voltage mobility

Length of the DNA moleculesmaller molecules generate less friction and so

move fasterEthidium bromide (stain) intercalated into DNA

decreases charge to mass ratio and so decreases mobility

General procedure

1. Casting of gel2. Loading of gel sample3. Electrophoresis4. Staining and visualization5. Downstream procedure

Factors affecting resolutionResolution = separation of fragmentsThe “higher” the resolution, the more space

between fragments of similar, but different, lengths

Resolution is affected byagarose typeagarose concentrationsalt concentration of buffer or sampleamount of DNA loaded in the samplevoltage

Linear carbohydrate polymer extracted from red seaweed , agarbiose

forms a porous matrix as it gelsshifts from random coil in solution to structure in

which chains are bundled into double helices

What is Agarose ?

% Agarose (w/v)

Size Range (kb prs) for Optimal Separation

0.5 2-30

0.75 0.7-20

1.0 0.5-10

1.5 0.2-3

2.0 0.1-2

Resolution of ds linear DNA fragments in agarose gels

1. 1%gels are common for many applications.2. Up to 3% can be used for separating very tiny fragments

but a vertical polyacrylamide gel is more appropriate in this case

Buffer Systems Remember, buffer systems include weak acids

and/or bases that do not dissociate completely.If ions resulting from dissociation are “removed,”

more weak acid and/or base will dissociate.Purposes of buffer

Keep solution at pH compatible with molecules being separated

Generate ions consistently tomaintain currentkeep resistance low

Both gel and the solution in the gel box are buffered.

Buffer Systems (cont’d)

Two commonly used buffers for routine agarose gel electrophoresisTAE, pH 8.0, ~50 mM - Tris, Acetate, EDTATBE, pH 8.0, ~50 mM - Tris, Borate, EDTA

Tris (T) is a weak base.Acetic (A) acid and boric (B) acid are weak acids.

Acetic acid is more completely ionized at pH 8.0 than is boric acid, so TBE has a high buffer capacity than TAE.

Non-denaturing agarose gel loading solutions

Compositiontracking dyes

are used to follow progress of electrophoresis

sometimes interfere with later visualization of DNA

a solute to increase density so that sample falls to

bottom of loading well with minimal dilution

solute examples: glycerol, Ficoll

Other gel types, with different purposes, use different loading solutions!

Voltage voltage, rate of migrationto increase the voltage

increase the setting on the power supplyincrease the resistance

decrease the gel thicknessdecrease the ion concentration

if voltage is too high, gel meltsas voltage is increased, large molecules migrate at a

rate proportionally faster than small molecules, solower voltages are better for resolving large

fragmentsbut the larger ds DNA fragments are always slower

than the smaller ones

Ethidium bromide staining

Binds to DNA by intercalation between stacked bases lies perpendicular to helical axismakes Van der Waals contacts with bases above and

belowAllows DNA visualization after gel electrophoresisEtBr intercalates with DNA and fluoresce under

ultraviolet light thereby allowing DNA visualization after Gel Electrophoresiswhile

Proteins may be visualised using silver stain or Coomassie Brilliant Blue

Agarose Gel Electrophoresis :OVERVIEW