ISOELECTRIC FOCUSING PPT.pptx

Isoelectric focusing is a technique used in biochemistry and molecular biology to separate and analyze proteins based on their isoelectric points (pI). It involves the use of an electric field to separate proteins in a gel matrix based on their pI values. Proteins migrate towards an electrode until they reach a pH region that corresponds to their pI, where they become electrically neutral and stop migrating. The technique is commonly used in protein purification and characterization, as well as in clinical diagnosis and research.

1. By Ananya Sharma B.sc(Honours) Forensic Science 2 nd Semester Enrollment number- 210160236009 ISOELECTRIC FOCUSING

2. Isoelectric focusing (IEF) Introduction Principle Procedure Applications

3. WHAT DO YOU MEAN BY ISOELECTRIC FOCUSSING.. Electrophoretic method that separates proteins according to differences in their isoelectric point (pI). Electrophoresis is the migration of charged molecules , particles or ion in a liquid medium under the influence of an electric field. Is ideal for separation of amphoteric substances.

4. Can use polyacrylamide or agarose gels Based on molecules that can be positively or negatively charged( amphoteric) For example: proteins, enzymes, peptides The net charge of protein is the sum of positive and negative charges of amino acid side chains When the net charge is zero that is the protein isoelectric point When a protein is reached its isoelectric point, it will stop migrating through the gel at a certain ph point .

5. Principle IEF works the principle on the property of individual protein molecule to stop movement at a certain pI under an electric field. But once the electric field is removed the molecules start to diffuse.

6. ISOELECTRIC POINT ( pI) The pH at which net charge on protein becomes zero Below pI – Positive charge Above pl – Negative charge Proteins move toward the electrode with the opposite charge During motion, proteins will pick or loose protons.

7. 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 are stripped from residue side chains. Proteins move in the electric field toward the distant cathode and away from the nearby anode. As the proteins move through the pH gradient, they gain positive charge and reach neutrality. At Ph=pl, the proteins have no charge and stop. Proteins stop exactly when Ph=pI and the stained proteins are very visible.

8. Separation is achieved by applying a potential difference across a gel that contain a pH gradient. Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel Isoelectric focusing gels contain synthetic buffets called ampholytes that smooth the pH gradient. Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids Commercially available ampholytes are- BIO-LYTE PHARMALYTE

9. Applications Widely used for separation and identification of serum proteins Used in food and agricultural industries, forensic and human genetic laboratories. Used in enzymology, immunology and membrane biochemistry. 2D Gel Electrophoresis is an application of IEF.