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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