isoelectric focusing (electrophoresis)

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Isoelectric Focusing (Electrophoresis): 

Isoelectric Focusing (Electrophoresis) By: Nirav Rathi M.Pharm(QA) S em-II BMCPER (MODASA)

Isoelectric Focusing: 

Isoelectric Focusing INTRODUCTION Principle Equipment Theoritical aspects Preparation Procedure Application Content: 2

INTRODUCTION:: 

INTRODUCTION : Also known as electrophoresis. Harry Rilbe and Tiselius were the major scientists to work on isoelectric focusing . Isoelectric focusing is a technique for seperating different biomolecules on the basis of their isoelectric points in a stabilized pH gradient . 3

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Net charge pH Isoelectric point NH 3 + COOH NH 3 + COOH pH < pI Positive charge NH 3 + COO - NH 3 + COO - pH = pI NH 2 COO - NH 2 COO - pH > pI Negative charge Isoelectric point (pI ): Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point. 4

Principle:: 

Principle: Protein is a mixture can be precipitated depending on its isoeletric point . IEF requires stable pH gradient which can be formed by using mixture of specially designed amphoteric molecules known as ampholytes. When electric field is applied, a pH gradient is established, that is negatively charged ampholytes moves towards anode and positively charged towards cathode and align themselves according to their pIs . 5

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When a protein has reached its isoelectric point, it will stop migrating through the gel at a certain pH point. 6

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Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution Traditional Equipment for Isoelectric focusing (IEF): 7

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IPGphor (IEF System) Amersham Pharmacia Biotech Inc. Protein IEF Cell Bio-Rad Laboratories 8

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Theoritical aspects:: 

Theoritical aspects: A protien at isoelectric point,its net charge is zero and can’t move in gel matrix. But it can move through diffusion. The pH gradient forces a protein to remain in its isoelectric point.Thus this concentrating effect is called “FOCUSING ”. The separation of bands is estimated by the minimum pI difference (∆ PI ). 11

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D Diffusion co.eff of protien d pH/ dx pH gradient E Intensity of electric field, in volts per centimeter. -dµ/dpH Variation of the solute mobility with the pH in the region close to the pI. The separation can by improved by narrowing pH range & by increasing the intensity of the electric field. ∆ PI = 3 x 12

Preparation:: 

Preparation: For polyacrylamide gel: 29.1g of acrylamide+ 0.9g of methylenebisacrylamide in 100 ml water. To 2.5 vol of this sol, add the mixture of ampholyte & dilute to 10 vol with water. Mix carefully and degas the sol. Then add the 0.25 vol of ammonium persulphate & Tetramethylethylenediamine Finally fill the moulds with the sol. 13

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Immobilized pH Gradient (IPG) Polyacrylamide gel Acidic buffering group: Basic buffering group: CH2 = C-HC-NH-R O COO - NH + Acrylamide monomer 14

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Gradient maker plastic support film A C B F E D acidic basic pH 3 pH 10 Production of Immobilized pH Gradient (IPG) strip 15

Procedure:: 

Procedure: Dismantle mould and the gel formed is transferred in to on to cooled support . Prepare the test and std sol and apply them on strip of paper which is placed above the gel. Cut the strip of gel & place them in electrolyte sol i.e acid for cathode and alkaline for anode. Supply the current and stop when the migration of proteins in std has stabilized. Some times the sample may be introduced directly in to column . 16

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Remove the sample and immerse it in 10%v/v Trichloroacetic acid for fixing and incubate for 30 min. Drain off the sol and add 200ml of destaining sol and shake it for 1 hr. Then finally add Coomassie Brilliant Blue or Crowle’s double stain. Destain the gel by passive diffusion untill the bands are visible . 18

Precautions:: 

Precautions: Samples should not be applied close to the electrodes. Cathodic shift i.e ( migration of protein to the cathode end of gel) may occur . Due to: If the gel is focused for more time the pH gradient decays. Due to release of Carbon dioxide. Salt content in samples are problematic, So, they are prepared in deionised water or 2% of ampholytes . 19

Application:: 

Application: IEF gel is used as identity test when migration pattern on gel is compared with std preparation. Used in limit test when the density of band is compared with the density of band of std prep. Used in quantitative estimation of conc of protein in the bands based on density using densitometer . 20

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