General Principles of Electrophoresis

Dr. Gangadhar Chatterjee23/07/2014

Electrophoresis

a separation technique Simple, rapid and highly sensitive

used in clinical laboratories to separate charged

molecules from each other in presence of electric field – Proteins in body fluids: serum, urine, CSF

– Proteins in erythrocytes: hemoglobin

– Nucleic acids: DNA, RNA

Clinical applications of Electrophoresis Serum Protein Electrophoresis

Lipoprotein Analysis

Diagnosis of Haemoglobinopathies and Haemoglobin A1c

Determination of Serum Protein Phenotypes and Micro heterogeneities eg. α1- antitrypsin deficiency, MM

Genotyping of Proteins eg. ApoE analysis for Alzheimer’s disease (polymorphic protein)

Small Molecules (Drugs, Steroids) Monitoring

Cerebrospinal Fluid Analysis

Urine Analysis ( determination of GNs)

Typical oligoclonal bands found only in CSF in Multiple sclerosis ( IEF & Western blotting)

Diagnosis of CJD by Immunoblot after SDS-PAGE separation of A) Human brain protein B) CSF of CJD patient C) Normal CSF

Where are the charges from?

Proteins

General Principles

An idealized, simplified situation: an isolated charged particle in a non conducting medium.

The force experienced by a particle in an electrical field is given by Coulomb’s law,

F = ZeE (E-electric field: potential per unit length) The viscous resistance of the medium to the motion:

-fv (f: the frictional factor) The viscous resistance of the medium just balances

the driving force. fv = F = ZeE

Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)

U = v/E = Ze/f

The zonal techniques: In these methods, a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix.

The kind of supporting matrix used depends on the type of molecules to be separated and on the desired basis for separation: charge, molecular weight, or both.

Almost all electrophoresis of biological macromolecules is at present carried out on either polyacrylamide or agarose gels

The matrix provides stability against convection and diffusion. In addition, in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size.

allows a permanent record of results through staining after run

high OH-High H+

What can we deduce from it?

Factors Affecting Electrophoresis

Interrelation of Resistance, Voltage, Current and Power

Two basic electrical equations are important in electrophoresis

The first is Ohm's Law, I = V/R The second is H = VI

( heat produced per unit time)

This can also be expressed as H = I2R

In electrophoresis, one electrical parameter, either current, voltage, or power, is always held constant

Ideally voltage is kept constant.WHY?

Under constant current conditions (velocity is directly proportional to current), the velocity of the molecules is maintained, but heat is generated.

Under constant voltage conditions, the velocity slows, but no additional heat is generated during the course of the run

Under constant power conditions, the velocity slows but heating is kept constant

Temperature and Electrophoresis

Important at every stage of electrophoresis

During Polymerization

- Exothermic Reaction

-Gel irregularities

-Pore size

During Electrophoresis

-Denaturation of proteins

-Smile effect

-Temperature Regulation of Buffers

Effect of matrix concentration

Agarose (%) Range of separation of linear DNA (in kilobases)

0.3 60 - 5

0.6 20 - 1

0.7 10 - 0.8

0.9 7 - 0.5

1.2 6 - 0.4

1.5 4 - 0.2

2.0 3 - 0.1

Electrophoresis Equipment: Horizontal or Submarine Gel

DNA/RNA is negatively charged: RUN TO RED

Agarose Gel Electrophoresis System

Homogeneous buffer

Discontinuous buffer

Continuous and Discontinuous Buffer Systems

A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel

In a discontinuous system a nonrestrictive large pore gel, called a stacking gel, is layered on top of a separating gel

The resolution obtainable in a discontinuous system is much greater than that obtainable in a continuous one. However, the continuous system is a little easier to set up.

Types of Electrophoresis

Agarose, cellulose, polyacrylamide

Iso-electric focusing

Capillary electrophoresis

Two-dimensional electrophoresis

Different stains of Electrophoresis

Plasma Proteins - Amido black

- Coomassie Brilliant Blue

- Bromophenol Blue

Hemoglobins

- Amido black

- Coomassie Brilliant Blue

- Ponceau Red

Lipoproteins

- Sudan Black

DNA ( Fluorescent dyes)- Ethidium Bromide- Sybr Green, Sybr Gold

Destaining solution used in most cases are Methanol and acetic acid mixture only.

Few technical considerations

What is EEO & why low???

Electroendosmosis cont

Normal patterns

Plasma protein HemoglobinAlkaline pH

DNANormal

Troubleshooting

Thanks