Dr. M. Siva Kumar Reddy MD

ELECTROPHORESIS

common Indications for electrophoresis

Multiple myeloma *a sharp distinct M band appears in -globulin fraction* Acute infections *α1 and α2 globulins are increased* Nephrotic syndrome *decreased albumin with sharp and prominent α2

globulin* primary immune deficiency *Diminished -globulin band* α1-Anti trypsin deficiency *diminished α1-globulin band*

Other indications in clinical chemistry

Waldenstrom’s macroglobulinemia Primary amyloidosis Unexplained peripheral neuropathy New onset anemia with renal failureHypercalcemia related to malignancyRouleaux formation on peripheral smear renal insufficiency with associated

serum protein elevation.

Unexplained pathologic fracture or lytic lesion on radiograph...

CNS infections and inflammationsDyslipidemiasEpidemics (micro organism causing

epidemic can be detected using PFGE)Drug monitoring using capillary

electrophoresis

Other indications in clinical chemistry

INTRODUCTION

Electrophoresis is a physical method analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes.

DEFINITION

Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electric field.

TYPES OF ELECTROPHORESIS

ELECTROPHORESIS

ZONE ELECTROPHORESIS

MOVING BOUNDARY ELECTROPHORESIS

ZONE ELECTROPHORESIS

PAPER ELECTROPH

ORESIS

WHATMANN FILTER PAPER ELECTROPHO

RESIS

GEL ELECTROPH

ORESIS

CAPILLARY ELECTROPH

ORESIS

GEL ELECTROPHORESIS

AGAROSE GEL ELECTROPHORESIS

POLYACRYLAMIDE GEL ELECTROPHORESIS

SDS-PAGE

DISC ELECTROPHORESIS

ISOELECTRIC FOCUSSING

IMMUNOELECTROPHORESIS

TWO DIMENSIONAL ELECTROPHORESIS

MOVING BOUNDARY ELECTROPHORESIS

NO SUPPORTING MEDIUM USED FOR MOVING BOUNDARY ELECTROPHORESIS

Arne Tiselius (10 August 1902 – 29 October1971)  Swedish biochemist 

ZONE ELECTROPHORESIS

Basic components of zone electrophoresis…..

supporting medium. Buffer chamber. Power pack and electrode. Sample dissolved in buffer tracking dye. Perspex cover to encase the entire

chamber.

Buffers

Most commonly used buffers in zone electrophoresis.

*ACETATE

*PYRIDINE

*BARBITONE

*TRIS(2-Amino,2-hydroxy methyl,1-3diol) most commonly used

*CITRATE

Buffers at a glance……..

Barbitone buffer(pH-8)• s.Protein

separation• Poor

resolution• Week buffer

Phosphate buffer(pH7)• Enzymes

separation• Low buffering

capacity• High

conductivity

TRIS- BORATE –EDTA(TBE)(pH-8)• Nucleic acid

separation• Good resolution

&high buffering capacity

• Low conductivity

Buffers at a glance………

TRIS-acetate-EDTA(pH-8) (TAE)• Nucleic acid

separation• High resolution &

high buffering capacity

• Low conductivity

TRIS- glycine buffer(pH >8)• Protein separation• High buffering capacity• Low conductivity

Preparation of Buffer

TRIS- GLYCINE buffer

In 600 ml

distilled water

3.6grams TRIS

4.6 grams

GLYCINE

PROCEDURE1 • Sample is spotted on supporting

medium

2 • Medium is placed between electrodes

3 • Power pack is switched on

4• Voltage applied till dye has

travelled approx 2cm from other end

5• Medium removed and dried• stained with coomassie brilliant

blue

6 • Destained with 5% acetic acid

7 • Bands are visualized and analyzed using densitometer

Factors influencing electrophoresis

Supporting

medium

Buffer

Electric

current

Size,shape &net charge of molecule

Heat and electroendosmosis

Paper electrophoresis

Paper electrophoresis is mainly used for separation of protein(at low voltage), amino acids and nucleotides(at high voltage).

Greatest disadvantage of paper electrophoresis is diffusion, adsorption and electroendosmosis.

To over come these issues high voltage is applied 10,000 volts that results in good resolution and rapid separation.

Small molecules like amino acids and nucleotides are separated by high voltage electrophoresis

Paper electrophoresis

Cellulose acetate Electrophoresis

General procedure is similar to that of paper electrophoresis

Cellulose acetate electrophoresis has replaced paper

electrophoresis

Better resolution in short time.

Negligible adsorption and EEO

Suitable for fractionation of polysaccharides and lipoproteins

Bands are easily detected due to transparency of medium.

Cellulose acetate Electrophoresis

Pulsed Field Gel electrophoresis

Separation of DNA using constant electric field is called constant field gel electrophoresis(CFGE).

Separation of DNA altering the strength and direction of electric field between the electrodes is called as pulse field gel electrophoresis(PGFE)

PGFE used to separate high molecular weight DNA.

CFGE is normal agarose gel electrophoresis for molecular weight up to 500kb.

PGFE used to separate human chromosome and yeast chromosome with sharper bands and high resolution.

PFGE

SDS PAGE….

Polyacrylamide gels are used for protein fractionation in the presence of SDS(sodium dodecyl sulphate)

Polyacrylamide could be used in the form of tube gel(for small volume of samples)

Vertical slab gel (for large volume of samples) Denaturing of protein sample is needed because protein structure is stable

with hydrophobic interaction, hydrogen bond, and disulphide bond. Stable structure does not favour electrophoresis and need to disturb its

structure Denaturing agents SDS, urea, and beta mercaptoethanol

polyacrylamide gel electrophoresis protein electrophoresis/ SDS PAGE

PROCESS OF SDS PAGE

Denatured

sample loaded

Running buffer(TRIS-

GLYCINE) ph8.8

80volts current

applied& allow it till

it reach 5cm above lower end

Staining by

coomassie or silver staining

Destaining using

methanol, acetic acid, water

mixture

SDS PAGE Applications

Determine molecular weight of proteinsTo fractionate protein subunitsTo assess the purity of proteins samples

Native PAGE with out SDS To separate enzymes and

isoenzymes

Disc electrophoresis (tube electrophoresis)

Disc electrophoresis(tube electrophoresis)

Tank of 100 ml buffer forms anode above and another tank of 100 ml forms cathode below.

Six holes of 2mm diameter are drilled1.5 cm apart and 1mm above the buffer.

Any protein specimen can be fractioned using this apparatus.

The order of resolution is prealbumin, albumin , postalbumins , ceruloplasmin ,transferrin, post transferrin fractions and gamma- globulins.

Lipoproteins separation and studies on Isoenzymes were also carried out using this apparatus.

Disc electrophoresis (tube electrophoresis)

Iso electric focusing

IEF is a type of gel electrophoresis in which compounds are separated in a gel with ph gradient , based on differences in iso electric point.

IEF gel has pre selected pH range.Compounds with pH less than iso electric pH

acquire negative charge and move towards anode and vice versa move towards cathode.

Migration continues till the molecules reach a pH region in the gel, which is equal to their iso electric point.

At that point molecules become stationary and exist as zwitter ions.

Isoelectric focusing

IEF applications

To determine iso electric point of protein.

To separate iso enzymes.

To fractionate proteins with higher resolution.

To separate all amphoteric substances.

To study mono,di,tri substituted derivatives of

proteins.

2D PAGE

In 2D PAGE, the molecules are first separated by IEF in a gradient gel, and then by SDS PAGE in a second gel.

2D PAGE IS USED: * To resolve proteins and enzymes with high resolution *To assess purity of proteins.

2D PAGE

Molecules separated by…• pH gradient

gel cast on narrow cylindrical tube

Gel extruded from tube…• Incubated in

SDS for 30 min to denature the protein

Placed on second gel and stacking

gel, voltage applied and

protein sub units are separated

Immunoelecrophoresis

Rocket electrophoresis

IEF combines sensitivity of gel electrophoresis and specificity of immune reaction.

This technique is used to quantify antigen.

Capillary electrophoresis In capillary electrophoresis, separation

is done with in capillary tube under high pressure and high voltage to give better resolution in short time

Capillary electrophoresis applications

To separate amino acids.

To separate oligonucleotides and nucleic acids.

To separate drugs.

To separate metal ions.

To separate enantiomers.

Agarose Gel Electrophoresis

Is used for fractionation of both DNA and protein

But with different buffers and at different current and voltages.

Agarose gel is used as supporting medium which is a polysaccharide derivative of agar.

The pores of agarose gel act as molecular sieve.

Pore size of gel is inversely proportional to the initial concentration of agarose

0.8% of agarose used for DNA of 0.5-10kb.

Process of gel electrophoresis

1% AGAROSE 100MG WEIGHED AND TAKEN IN TO TEST TUBE

To the agar add 10 ml of TRISS-GLYCINE ……..

Place the test tube in boiling water bath for about 10minutes

After taking out from water bath evenly mixed agarose gel taken in to pipette…….

Pour evenly on to the slide using pipette

Serum+ bromophenol blue

Place the sample on the slide

POUR THE BUFFER IN TO THE TANK and connect wicks

Run power pack for 1.30 hours at 150volts for better results

Fixative (methanol or acetic acid)

For a period of 30 minutes leave the slide in the fixative

Drying of slide

Can use drier or hot air ovenDry the slide till the agarose fixes to slide

Preparation of COOMASSIE BLUE dye

Made to 100ml (CBB dye)

Glacial acetic

acid 7ml

Dye 300mg

Ethanol 50ml

Staining with coomassie blue

Leave the slide in the CBB stain for 30 minutes

Distaining with 5% acetic acid

Examine the slide for the bands

Normal serum pattern

Nephrotic syndrome

Analyze the slides using densitometer

Normal eletrophoretic pattern…

Inflammation vs normal

Normal vs multiple myeloma

Lipoprotein electrophoretic pattern…..

Hemoglobin electrophoretic abnormal patterns….

CSF Electrophoresis bands

Serum protein electrophoresis vs urine protein electrophoresis

Analysis of patterns of albumin………

Increased albumin Decreased albumin

• dehydration • Chronic cachectic or wasting diseases

• Chronic infections

• hemorrhage

• burns• Protein loosing

enteropathies• Impaired liver function

• malnutrition

• Nephrotic syndrome

Analysis of patterns of α1 globulins..

Increased α1 globulins Decreased α1 globulins

pregnancy α 1 antitrypsin deficiency

Analysis of patterns of α2 globulins ……… Increased α2 globulins Decreased α2 globulins

Adrenal insufficiency malnutritionAdrenocorticosteroid therapy

Megaloblastic anaemia

Diabetes mellitus Protein losing enteropathy

Nephrotic syndrome Severe liver diseaseWilson’s disease

Abnormal patterns of β1 or β2 globulins..

increasedβ1 or β2 globulins

decreasedβ1 or β2 globulins

Biliary cirrhosis Protein malnutritioncarcinomasCushings diseaseDiabetes mellitushypothyroidismIron deficiency anemiaMalignant hypertensionnephrosisPoly arteritisObstructive jaundice

Abnormal pattern of -globulins…… Increased -globulins

Decreased -globulins

amloidosis Agamma globulinemia Chronic infections hypogammaglobulinemia Chronic lymphocytic luekemia cirrhosis Hodgkin’s diseases Malignant lymphoma Multiple myeloma Connective tissue disorders Waldenstrom’s macroglobulinemia