Isoelectric focusing electrophoresis.pptx

Isoelectric focusing electrophoresis
Isoelectric-focusing electrophoresis is a type of electrophoresis. The separation technique involves electrophoresis based on the isoelectric point of the sample.

1. Isoelectric focusing electrophoresis Nishanth K P 1 st Semester M. Pharm (Pharmaceutics) Nazareth College of Pharmacy 1

2. INTRODUCTION IEF electrophoresis is began in 1964 In isoelectric Iso means ‘same’ and Electric means ‘charge’ Iso electric focusing (IEF), is a technique for separating different molecules by based on their isoelectric point The isoelectric point is the pH at which the net charge of the protein is Zero. IEF is often used as part of the quality control testing of therapeutic biological products to demonstrate batch consistency. 2

3. PRINCIPLE IEF is performed in a pH gradient Proteins are amphoteric molecules with acidic and basic buffering groups In basic environment, the acidic groups become negatively charged In acidic environment, the basic groups becomes positively charged Isoelectric point (pI) : The pH where the charge of a protein is zero Proteins with same molecular weights will separate out by pH 3

4. Proteins are positively charged in solutions at pH values below pI and migrate towards cathode Proteins are negatively charged in solutions at pH values above pI and migrate towards anode 4

5. All proteins have an isoelectric pH When the electrophoresis is run in a solution buffered at constant pH, Proteins having a net charge will migrate towards the opposite electrode so long as the current flows. The use of pH gradient across the supporting medium causes each protein to migrate to an area of specific pH. The pH of the protein equals the pH of the gradient, thus resulting in sharp well defined protein bands. A procedure to determine the isoelectric point (pI) of proteins thus, a mixture of proteins can be electrophorized through a solution having a stable pH gradient in from the anode to the cathode and a each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric focusing IEF is also known as Electrofocusing 5

6. PROCEDURE Protein is loaded at the top of a column where pH is very high Most of them are negatively charged at this pH Proteins move in the electric field towards the distant cathode and away from the nearby anode As the protein move through the pH gradient, they gain positive charge and reach neutrality At pH = pI, the proteins have no charge and stop Proteins stop exactly when pH = pI and the stained proteins are very visible Highly stable ampholytes are molecules with specific pKa to give a specific and unchanging pH gradient 6

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8. Separation is achieved by applying a potential difference across a gel that contains a pH gradient Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel Isoelectric focusing gels contains synthetic buffers called ampholytes that smooth the pH gradients Ampholytes are complex mixtures of synthetic poly amino poly carboxylic acids Commercially available ampholytes are; -BIO-LYTE -PHARMALYTE 8

9. Advantages IEF, greatest advantage is its high resolution, resulting in greater separation of solutes. IEF of serum proteins results in many more bands; these bands are sharper because each pH region is very narrow Performing IEF is easier because the placement of sample application is not important. The sample and ampholytes can be mixed before application, The ampholytes will migrate, create the gradient and then the proteins separate and migrate 9

10. APPLICATIONS IEF is a highly sensitive analytical technique and is particularly useful for studying microheterogeneity in a protein The method is useful for separating and identification of isoenzymes (Which are different forms of the same enzyme often differing by only one or two amino acid residues) 2D gel electrophoresis is an application of IEF. Protein is first separated based on pI and then based on molecular weight using SDS-PAGE Widely used for separation and identification of serum proteins Protein purification To measure pI values Immuno blotting assays 10

11. REFERENCES Friedman DB, Hoving S, Westermeier R. Isoelectric focusing and two-dimensional gel electrophoresis. Methods in enzymology. 2009 Jan 1;463:515-40. https://www.excedr.com/resources/isoelectric-focusing-electrophoresis-overview https://www.slideshare.net/arushe143/isoelectric-focussing 11

12. Thank You 12