Iso electric focussing gel electrophoresis

Dr Anurag yadav,Bio-FMMC

Presenter Dr Anurag YadavPost-graduate, biochemistry

Father Muller Medical college

1

ELECTROPHORESIS

Content :

Dr Anurag yadav,Bio-FMMC2

Isoelectric focussing gel electrophoresis

Two dimensional electrophoresis

Isotachophoresis

Pulsed field electrophoresis

High voltage electrophoresis

Isoelectric focussing gels


First described by- H.Svensson in Sweden.Method is ideal for the separation of the

amphoteric substances.Method has high resolution.Able to separate the protein which differ in

isoelectric point by little 0.01 of pH unit.Most widely used as the horizontal gel slab.


Different gradient of the pH along the length of the separating gel.

Establishment of ph gradient:


This is achieved by the ampholyte & must have following prop:Must dictate pH course (buffering capacity at their Ip)Should have conductance at their Ip.Low molecular weightSoluble in waterLow light absorbance at 280nm.

Available commercially with pH band (3-11)Eg: Ampholine, Pharmalyte and Bio-lyte.


Movement



Duration : 2-3hHigh voltage : 2500VCooling plates : 100CStable power packFixing (trichloroacetic acid) and Staining

(Coomassie Brilliant blue)


Application:- Highly sensitive for studying the

microheterogeneity of proteins- Useful for separating the isoenzymes.- Human genetic lab- Research in enzymology, immunology,- Forensic, food and agriculture industry,

Two-dimensional polyacrylamide gel

electrophoresis


Principle :

Technique combines with IEF as first dimensional.• Which separate

according to the charge.

Second dimension by SDS-PAGE• Separate according

molecular size.


Thus combination gives sophisticated analytical method for analysing the protein mixture.

Size very from 20*20cm to the minigel.

IFE is carried on acrylamide gel (18cm*3mm), with 8M urea.

After separation, placed on 10% SDS-PAGE for further separation .


Used in field of proteomics.Can separate 1000 to 3000 proteins from

the cell or an tissue extract.


Isotachophoresis


Used for separation of smaller ionic substances.

They migrate adjacent with contact one another, but not overlapping.

The sample is not mixed with the buffer prior to run.

Hence current flow is carried entirely by the sample ions.

Faster moving ions migrate first and the adjacent ones next with no gap between the zone .


All ions migrate at the rate of fastest ion in zones.

Then it is measured by UV absorbance.Application-

Separation of small anions and cationsAmino acidsPeptidesNucleotidesNucleosidesProteins.

Pulsed-Field Electrophoresis


Power is applied alternatively to different pair of electrodes

Electrophoretic field is cycled at 105-1800

Because of which the molecule have to orient to the new field direction

This permit separation of large molecule like DNA .

Applied: for typing various strain DNA.

High voltage electrophoresis


First described by Michl.As the name describe, the electrophoresis

is carried under the very high voltage.This is required for the substances of lower

molecular weight which will have considerable high diffusion rate.Eg: amino acids, peptides.


The voltage applied was ranging from2500-10000 V or 50-200V/cm, 500mA.This resulted in better resolution and even

very rapid separation.And even with tremendous amount of heat

generation.To tackle this, it need a good cooling

system.

components


Buffer reservoirsCooling platePressure padelectrodesPower sourceInsulated coverWickRefrigerating unit


Precautions :Temperature of the system has to be maintained constant.

Plate dimension 50*50cmThe HVE one direction can be combined

with the chromatography- which is right angle to first.

Possible even to run in two direction at two different pH.


THANK YOU

References


Keith Wilson- Principles and techniques of biochemistry and molecular biology.

Upadhyay- biophysical chemistry.Tietz- Text book of clinical chemistry.Kaplan- clinical chemistry.YouTube and Google images.

Health & Medicine

Iso electric focussing gel electrophoresis