Electrophoresis

• Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge

• As such, it is one of the most widely-used techniques in Hematology ,biochemistry and molecular biology

• When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.

Electrophoresis is carried out by applying a thin layer

Solid matrix with pores which are used:• paper• starch• cellulose acetate• polyacrylamide• agar/agarose

Molecules in the sample move through porous matrix at different velocity.

Haemoglobin Electrophoresis

• Haemoglobin Electrophoresis is a method of determining the type and quantity of haemoglobin in blood, by observing the rates of transit of these negatively-charged proteins in an electric field medium. It is used to diagnose so-called haemoglobinopathies

• The most common types of normal hemoglobin are:• Hemoglobin A.(α2β 2)This is the most common type of

hemoglobin found normally in adults• Hemoglobin F (fetal hemoglobin (α2γ2 )This type is

normally found in fetuses and newborn babies. Hemoglobin F is replaced by hemoglobin A (adult hemoglobin).

• It is increased in Thalassemia Major• Hemoglobin A2(α2δ2) This is a normal type of hemoglobin

found in small amounts in adults.• It is increased in Thalassemia Minor

Abnormal hemoglobin. 1

• There are more than 350 types of abnormal hemoglobin. 1 The most common are:

• Hemoglobin S. This type of hemoglobin is present in sickle cell disease.

• Hemoglobin C. This type of hemoglobin does not carry oxygen well.( Hemoglobin C disease)

• Hemoglobin E. This type of hemoglobin is found in people of Southeast Asian descent.(bengali)

• Hemoglobin D..(Punjabi)

Normal Haemoglobin• Hb A: 95% to 98%• Hb A2: 2% to 3%• Hb F: 0.8% to 2%• Hb S: 0%• Hb C: 0%• In infants and children, these hemoglobin molecules make

up the following percentages of total hemoglobin:• Hb F (newborn): 50% to 80%• Hb F (6 months): 8%• Hb F (over 6 months): 1% to 2%

Electrophoresis is carried out by applying a thin layer

Solid matrix with pores which are used:• paper• starch• cellulose acetate• polyacrylamide• agar/agarose

Molecules in the sample move through porous matrix at different velocity.

Cellulose acetate electrophoresis

• Cellulose acetate is an acetate salt of cellulose produced by treating cotton with acetic acid using sulphuric acid as a catalyst.

• Migration takes place on the buffer film on the surface of the cellulose acetate plate or membrane.

• Cellulose acetate electrophoresis may be used for qualitative identification of variants, but also with elution for quantization of the haemoglobins, A2, A, S, D, Lepore, α-chain variants, Hb H and Hb Bart's.

Reagents and materials • Tris-EDTA Boric Acid (TEB) buffer, pH 8.4. -Tris hydroxymethyl amino methane (TRIS): 10.2 g -Ethylene diamine tetracetic acid (EDTA): 0.6 g -Boric Acid: 3.2 g -Make up to 1 litre with distilled water • Whatman No. 3 chromatography paper. • Cellulose acetate membranes made by Sartorius and by Shandon • HbA2 control, as supplied by the National Institute for Biological Standards and

Control NIBSC. • The control has been produced by freeze drying a solution of haemoglobin

prepared from human cells and made stable by the addition of sucrose (200 mM), potassium cyanide (6mM) and chloramphenicol (1 mg/dl). Reagent with an assigned value of 5.3% of total haemoglobins present.

Haemolyzing reagent CCl4

Fixative/stain solution• Ponceau S- 5 gms• Trichloracetic acid 7.5 gms• Water 1 litre Destaining solution 3%(v/v) acetic acid and 30 ml water and Make to 1 litre

Equipments

• Power supply capable of delivering a constant current

• An horizontal electrophoresis tank with adjustable bridge gaps and a polarity indicator (eg. Shandon).

• Roller mixer. • Spectrophotometer

Haemolysate preparation

• Collect blood in 5 ml blood in EDTA vial• Wash RBC three times• Lyse 2 volumes of washed packed RBC in one Volume of distt. water and then add CCl4 Or lyse By freezing and thawing the add CCL4.• Shake the tubes vigorously for approxim for 1 mt.• Centrifuge at 3000 rev/minute) for 30 mts.• Transfer the supernatant to a clean tube adjust• Hb to 10 gms/dl

Method

• The electrophoresis tank is prepared by filling

the tank with 900ml approximately of TEB buffer wicks are cut from Grade No. 3 chromatography paper and were placed along the 22 cm long bridges in the tank.

• The cellulose acetate membranes are cut in 40xl00 mm each and soaked (shiny side down) in TEB buffer for 5 minutes. Five strips are plotted and placed on the electrophoresis tanks.

• Voltage current is applied at 250 V for 5 minutes to the membranes to equilibrate the membranes with the buffer.

• The current is turned off and 5-10 μl haemolysate (10 g/dl) is applied on each membrane at the cathodal end using a capillary tube.

• Then the voltage is set at 250-300 V working at constant current of 2 mA for each strip.

• The electrophoresis is run for approximately 45 minutes to one hour until there is a clear area between the bands.

• After 25 mts electrophoresis,immediately transfer cellulose acetate membrane to

• Ponceau S for 5 min.• Remove excess stain by washing for 5 min. in First acetic acid reservoir and for 10 mts in

each of remaining two.

• The current is then turned off and the separated HbA2 on the cellulose acetate membrane is cut and immersed in a tube containing 4 ml of distilled water and the HbA in a tube containing 16ml distilled water. If a haemoglobin variant is present then this is cut separately into 4 or 16 ml distilled water depending on the quantity of the variant present.

• Note that a blank is prepared from the same run by cutting a piece of clear cellulose acetate strip that was immersed in 4ml distilled water.

• The tubes are then placed on a roller mixer for 30 minutes for the haemoglobin elution.

• The strips are removed and the tubes are then centrifuged for 10 minutes at 3.000 rpm.

• The absorbance of each haemoglobin is read at 413 nm against the blank on a spectrophotometer.

• The results are calculated as follows: Absorbance of HbA2 x 100

• % of HbA2 = --------------------------------------- Abs. of Total (HbA x 4) + absorbance of HbA2

• The haemoglobins migrate on the cellulose acetate membrane from cathode to anode in the following order: HbA2, Hb E, Hb C, Hb D, Hb S, Hb Lepore, Hb F, Hb A and the fast moving haemoglobins Bart's and Hb H

Cellulose acetate electrophoresis Jump to: navigation, search

Cellulose acetate is an acetate salt of cellulose produced by treating cotton with acetic acid using sulphuric acid as a catalyst. Migration takes place on the buffer film on the surface of the cellulose acetate plate or membrane. Separation of the proteins is primarily by charge. Cellulose acetate electrophoresis may be used for qualitative identification of variants, but also with elution for quantitation of the haemoglobins, A2, A, S, D, Lepore, α-chain variants, Hb H and Hb Bart's.

• Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional.

• One dimensional electrophoresis is used for most routine protein and nucleic acid separations. Two dimensional separation of proteins is used for finger printing , and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).

• Most common stabilizing media are polyacrylamide or agarose gels.

Support media:

1. Filter paper: paper electrophoresis (Principle same as paper electrophoresis)

Moist paper is placed on a supporting rack, samples are applied with a capillary applicator, paper is loaded onto the apparatus with its ends dipping into an electrode vessel containing buffer, and covered to prevent dryingat the end of run, paper is

carefully taken out and dried, and examined by appropriate detection technique

Disadvantage

• Thin paper: Tear

• Thick paper: Boundaries between the zones distort

• OH group of cellulose interact with biomolecules and results in ‘tailing’

• water absorbing and electrical conductivity capacity of paper depends on its content (96% -cellulose)

• Electro osmosis

Electro osmosis• dissociation of COOH group(cellulose matrix)

generation of (H3O)+ ionMoves (electric field)• A correction factor for electrophoretic mobility: • distance between the sample origin and the position

of the blue dextran (does not ionize)• 20V cm-1 – zone diffusion of small molecules• 200 V cm-1 – High voltage EP

2. Cellulose Acetate Membrane: Advantages over paper

• OH groups of cellulose are esterified by acetylation

• spectrophotometric determination:Use of mineral oil of RI equals to that of the membrane;

• Easy dissolution: Recovery of separated sample• Procedure similar to paper

3. Gel Media:

Network of polymer molecules

surrounded and penetrated by buffer

space between the gel molecules are the porous

frictional resistance to movement

(1) relative size of gel pore

(2)radius of the molecule;

exerts a sieving effect

a) Starch Gels:

• earlier work with proteins used starch as support• cheap, easy and reasonably quick

Disadvantage• pore size can vary within only a narrow range

(determined by the starch conc)• contains some negatively charged side-chain:

hinder separation

b) Polyacrylamide Gels:

Made by cross linking acrylamide with N, N’-methylenebisacrylamide

Requires an initiator (APS of PPS) for chemical polymerization & light and ribofalvin for photopolymerization

Crosslinking reagent is N, N, N’, N’-tetramethylethylenediamine (TEMED)

Ratio of cross-linking agent to acrylamide is a factor determines the pore size of the gel

Can be tube gel or slab gel

1. Tube Gel

2. Slab Gel EP: Advantage; heat dissipation is better, higher resolving power; can be used horizontally (when gel conc is low) or vertically

c) Agarose Gel:

Agarose, obtain from seaweeds, linear polymer of D-Gal and 3,6-anhydroGal

agarose dissolved in boiling water when cooled forms a gel (held by H-bonds),

pore size relatively large: large proteins or nucleic acids (too large to enter PAG)

Contaminants charge groups (sulphate and carboxylic acid ) interfere with separation

Factors affecting electrophoretic mobility:

Number of parameter of electrophoretic system:

i) for zone EP, type of support medium chosen, and if it is a gel, its pore size

ii) pH of the electrophoresis buffer

iii) ionic composition of buffer

iv) applied voltage

v) temperature

USES OF ELECTROPHORESIS

1. DNA analysisForensic investigationPaternity tests

2. protein analysisOf blood or urine samples

3. antibiotic analysisSynthesis new antibiotic Analyse which type of bacteria is antibiotic resistant

4. Vaccine analysis.