ISO ELECTRIC FOCUSING(ANALYSIS).pptx

soelectric focusing (IEF) is a technique used to separate molecules based on their isoelectric point (pI), which is the pH at which a molecule has no net charge. In IEF, a mixture of charged molecules, such as proteins or peptides, is applied to a gel matrix containing a pH gradient. An electric field is then applied across the gel, causing the charged molecules to migrate to their isoelectric point, where they become electrically neutral and stop moving.

The pH gradient in the gel can be created using different methods, such as immobilized pH gradient (IPG) strips or carrier ampholytes. IPG strips are precast gels that contain a pH gradient formed by covalently linking a series of buffering molecules of different pK values to the gel matrix. Carrier ampholytes, on the other hand, are small molecules that migrate in the electric field and create a pH gradient by buffering the surrounding solution.

During IEF, the charged molecules move through the pH gradient in the gel, reaching their isoelectric point and becoming immobilized at that point. The separation of the molecules is based on their differences in pI values, with molecules with higher pI values moving towards the anode and those with lower pI values moving towards the cathode. The separation is visualized by staining the gel with a suitable dye or by transferring the separated molecules onto a membrane for further analysis.

IEF has many applications in protein analysis and purification, including the separation of protein isoforms, identification of post-translational modifications, and characterization of protein-protein interactions. It is a powerful tool for identifying and characterizing proteins in complex mixtures and is commonly used in proteomics research.

1. SUBMITTED BY :- MOHAMMAD JAVED M.PHARM 1 ST SEM PHARMACEUTICAL CHEMISTRY SUBJECT:- MODERN PHARMACEUTICAL ANALYTICAL TECHNIQUE TOPIC :- ISO-ELECTRIC FOCUSING.

2. CONTENTS Introduction Graphical representation Principle Instrumentation Factor affecting seperation Advantage and disadvantage Summary Reference

3. INTRODUCTION It was first discovered by H.Sevennson , Sweden.(1) We use this technique as protein separation on the basis of Iso-elelectric point( p.I ). It is also called electro-focusing. It is a technique for separating different molecule in their iso -electric point. It is a type of zone electrophoresis performed by protein.(2) Iso -electric point( pI ) :- The pH at which net electric charge on protein becomes zero. Below p.I - positive charge(acidic) Above p.I - negative charge(basic) Protein moves towards the electrode with opposite charge.

4. Graphical representation of iso - electric point of Alanine

5. PRINCIPLE It is performed in pH gradient. The proteins are amphoteric molecules with acidic and basic buffering group. In acidic environment the basic group becomes positively charged while in Basic environment the acidic group becomes negatively charged . Proteins are positively charged in solution at pH value below pI,migrate towards cathode. Proteins are negatively charged in solution at pH value above pI , migrate towards anode . At pH= pI the protein have no charge and stop.(3)

6. INSTRUMENTATION Focusing Tray : These are the trays that hold the gel media, i.e. IPG strips on which proteins will migrate. IPG Strip : IPG(immobilized pH gradient) Strips are commercially available gel media having a stable pH gradient of mostly 3 - 10. These are formed by casting polyacrylamide gels using acrylamide buffers on a plastic backing. These are more stable and reproducible . Wick : Wicks are placed over the ends of the gel to collect salts and proteins/peptides that are outside of the pI range of the IPG strip . Electrodes : There are two electrodes of opposite charge are attached at the opposite end of the focusing tray which will provide the necessary electric field. Cup Holder : Samples are mixed with ampholytes and loaded in the Cups so that it can migrate.(2)

7. FACTOR AFFECTING SEPERATION pH Intensity of electric field Temperature Starting and find voltage Time Mode of ramping

8. ADVANTAGE AND DISADVANTAGE Advantages : Proteins that as by little 0.001 pH unit can be separated. I.E.F is a powerful analytical tool for separation of protein. Disadvantage :- Inconsistency Limited stability of solution Inadequate purity of application as a standard.

9. SUMMARY Food and agricultural industries. In enzymology, immunology, membrane bio chemistry. Forensic and human genetic lab. I.E.F is the 1 st step in 2D gel electrophoresis and then furthur separated by SDS-PAGE. It is used to separate different protein/peptide molecule on the basis of the pI and charge value .

10. REFERENCE Rilbe , H. (1973, June). HISTORICAL AND THEORETICAL ASPECTS OF ISOELECTRIC FOCUSING. Annals of the New York Academy of Sciences , 209 (1 Isoelectric F), 11–22. Mahadik k, Sathiyanarayan L:, Instrumental Method of analysis: Nirali publication, August 2020; Page no,: 13.4.3 Pergande, M., & Cologna , S. (2017, January 25). Isoelectric Point Separations of Peptides and Proteins. Proteomes , 5 (4), 4. https://doi.org/10.3390/proteomes5010004