Electrophoresis

 “Separation of macromolecules on the basis

of difference in charge-density and molecular
weight”

 “Migration of charged molecules in response

to an electric field”.

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Molecular Migration

 Strength of the electric field

 Charge
 Size
 Shape of the molecules
 Ionic strength
 Viscosity
 Temperature of the medium in which the
molecules are moving.

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Analytical Tool
 Relatively inexpensive
 Simple,
 Rapid
 Highly sensitive.
 Proteins, peptides, amino acids
 Nucleic acids, nucleotides

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Theory of Electrophoresis
 v=Eq/f
v= velocity, E=electric field,
q=net charge, f=frictional co-efficient.
 µ=v/E
µ= mobility of a molecule
 µ=Eq/Ef
 µ=q/f
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 Methods of Electrophoresis

 Supporting medium
Cellulose
Thin gels
 Geometrics
Vertical
Horizontal
 Slab or column
 Buffers
 Electrophoresis conditions
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Types of Electrophoresis

 Agarose Gel Electrophoresis

 (Used to resolve Nucleic Acids)

 Polyacryamide Gel Electrophoresis

 (Used to resolve Proteins)

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Step involved in Protein
Electrophoresis

 Extraction of Proteins
 Determination of total protein concentration
 Preparation of sample
 Polyacrylamide gel electrophoresis (PAGE)
 Staining of gel
 Gel documentation
 Further analysis

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Extraction of Proteins

Buffers System

Some commonly used buffers are:

 Borate Buffer
 Tris Buffer

 PBS Buffer (phosphate buffer

saline)
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Types of PAGE

 Native PAGE (non-denaturing PAGE)

 The proteins are resolved in the absence of
SDS

 SDS-PAGE (Denaturing PAGE)

 The proteins are resolved in the presence of
SDS
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 +ive Features of PAGE

 High resolving power.

 Minimal interaction b/w migrating molecules
& matrix.
 Physical stability of matrix.
 Separation basis
Molecular sieving
Electrophoresis mobility

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 Gel Formation

 Acrylamide
 Bis-acrylamide
 Ammonium per sulphate (APS, initiator)
 TEMED (Tetramethylethylenediamine, catalyst)

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 Acrylamide
.

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Models of PAGE and Setups

Tube Gel

Slab Gel

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 Native PAGE

Native PAGE is used

to determine the
purity of a protein
or enzyme.

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What is SDS-PAGE?


Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis

A procedure to separate proteins and

determine their Molecular Mass

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What is so special about SDS?

 SDS is a negatively charged detergent

 Disrupts secondary and tertiary protein

structures by breaking hydrogen bonds

and unfolding protein
 ‘Masks’ charge on protein so that all

proteins act the same as regards

charge have same charge
 Prevents protein aggregation

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What Happens?

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Recipe of Solution
 Stock acrylamide bis Solution(30+0.8)g
 Resolving gel buffer: 3 M Tris-HCl pH 8.8.
 Stacking Gel buffer:0.5 M in Tris-HCl pH
6.8.
 APS: 1.5%
 SDS: 10%
 PAGE buffer: 0.25 M Tris-HCl, 1.92 M
Glycine, 0.1% SDS. pH 8.3

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Composition of Resolving gel

 Composition depends on final acrylamide conc.

Reagent: Volume (30 mL)

 Acrylamide/Bis Solution 10
 Resolving gel buffer (8.8) 3.75
 10% SDS 0.3
 H2O 14.45
 10 % NH4 persulphate 1.5
 TEMED (added last) 15 L

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Composition of Stacking gel

Reagent: Volume (20 mL)

 Stacking gel buffer (6.8) 2.50
 30 % acrylamide 2.50
 10% SDS 0.30
 H2O 11.3
 1.5 % NH4 persulphate 1.0 mL
 TEMED (added last) 15 

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 Staining of proteins in gels

1. Using Coomassie brilliant blue with G-250 (or

PAGE blue) followed by destaining

2. Silver staining requires a number of finicky

treatment and washing steps and takes at
least a couple of hours

 Silver staining is (or can be) extremely

sensitive - to 1 ng/band) than other stain
(Coomassie brilliant blue G-250)
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Staining of Gels Using Dye

Coomassie Brilliant Blue Staining

 Methanol: 40%
 Acetic acid: 10%
 Dye: 0.25%
 Water: 50%
 Filter prior to use.

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Destaining of Gels

 Methanol: 40%
 Acetic acid: 10%
 Water: 50%
 Filter prior to use.

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 SDS-PAGE analysis for seed proteins

P1 P2 P3 P2 P1 M
Invitrogen
10% SDS-P.A. Gel Protein ladder:
10064-012

60 kDa
50 kDa
40 kDa
30 kDa

20 kDa

10 kDa

P1 Hygrophila auriculata, P2 Abrus precatorius P3 Moringa oleifera P4 Croton tiglium

P5 Withania somnifera
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Protein ladders

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 Determination of Molecular Weight

 Known molecular weight along with the protein to be

characterized.
 A linear relationship exists between the logarithm of
the molecular weight of a PAGE resolved polypeptide
and its Rf.
 The Rf is calculated as the ratio of the distance
migrated by the molecule to that migrated by a
marker dye-front.

 Standard curve of distance migrated vs. log10MW for

known samples, and read off the logMr of the sample
after measuring distance migrated on the same gel.

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 Standard curve for Molecular Mass (kDa)
Determination

2 y = -1.8279x + 2.224
R2 = 0.9983
log of Mol. wt.

1.5

0.5

0
0 0.2 0.4 0.6 0.8
Rf values

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Western Blotting

 SDS-PAGE
 Transfer
 Western blot

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Protein Databases

 Swiss-Prot
 NCBI
 SEQUEST
 BLAST

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Agarose Gel Electrophoresis

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 DNA is negatively charged (because of
phosphate backbone)
 DNA will be attracted to positively
charged poles and repelled from
negatively charged ones

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Running a gel
 Molten agarose is poured into a casting tray and a
comb is placed
 After the agarose solidifies, the comb is removed
leaving wells where the DNA will be loaded
 DNA samples are mixed with tracking dye which
contains sucrose (to weigh down the DNA) and dyes
so that you can visualize migration
 A buffer containing ions (to conduct an electric
current) is placed in the chamber around the gel

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 Gel electrophoresis

- electrode DNA fragments + electrode

Agarose gel
~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

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 Movement of DNA fragments
in agarose gels

 There is a linear relationship between

the migration rate of a given DNA
fragment and the logarithm of its size
(in basepairs).
 Larger molecules move more slowly
through the gel because of more
friction

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Visualizing DNA
 Ethidium bromide
 A fluorescent dye visualized
when excited by UV light
 Intercalates into the DNA

molecule, thereby “staining” it

 Gel is soaked in a solution of EtBr and the
DNA bands take up the dye
 Then the gel is placed under UV light and
visualized and/or photographed

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An ethidium-stained gel photographed
under UV light

**Each band that you see is a collection of millions of

DNA molecules, all of the same length!!
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RNA ladders

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DNA ladders

 
 
 
 
 
 
 
 
 
 
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Thanks

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