Isoelectric focusing ppt.

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1. I S F COLLEGE OF PHARMACY MOGA ISOELECTRIC FOCUSING PRESENTED BY: LOVEINDER BHARDWAJ (PHARMACOLOGY)

2. Content INTRODUCTION ISOELECTRIC POINT PRINCIPLE AMPHOLYTE PROCEDURE SAMPLE PREPARATION AND LOADING CALCULATING PH APPLICATIONS

3. INTRODUCTION Isoelectric focusing (IEF), is a technique for separating different molecules by based on their isoelectric point. It is also known as electro focusing. IEF is performed in a ph gradient. Basic principle involved is electrophoresis IEF is well established as an excellent technique for the analysis of proteins, such as enzymes, hormones or other biologically active proteins.

4. ISOELECTRIC POINT The ph. at which net charge on protein becomes zero. Proteins are separated in a pH gradient according to their isoelectric points. Proteins are amphoteric molecules with acidic and basic buffering groups (side chain). 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.

5. PRINCIPLE Separates proteins by their isoelectric points. Each protein has own pl=ph at which the protein has equal amount of positive and negative charges i.e the net charge is zero.

6. AMPHOLYTE Ampholytes are low molecular weight molecules that help in creating a stable pH gradient. They are isomers of polycarboxylic acids. PROPERTIES: It should be soluble in water. It should have low absorption spectra. It should offer conductance.

7. Synthetic carrier ampholyte: High buffering capacity and solubility at the pI. Good and regular electric conductivity at the pI. Absence of biological effects. Low molecular weight. Natural occurring ampholyte: Amino acids or peptides Lack the properties above Can not be used in IEF.

8. What is a gel ? Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight and porosity of the target molecules. Types of Gel: Agarose gel. Polyacrylamide gel.

9. AGAROSE GEL A highly purified uncharged polysaccharide derived from agar. Used to separate macromolecules such as nucleic acids, large proteins and protein complexes. It is prepared by dissolving 0.5% agarose in boiling water and allowing it to cool to 40°C. It is fragile because of the formation of weak hydrogen bonds and hydrophobic bonds. Nontoxic gel medium Good for separating large DNA molecules and poor for separating small DNA molecules.

10. POLYACRYLAMIDE GEL Used to separate most proteins and small oligonucleotides because of the presence of small pores. Polyacrylamide gels are tougher than agarose gels. Polyacrylamide gels are composed of chains of polymerized acrylamide. Sharp bands.

11. PREPARATION AND SAMPLING ACRYLAMIDE, AMMONIUM PERSULPHATE(APS), TEMED, CARRIER AMPHOLYTE, UREA, WATER. An electrophoresis chamber and power supply Gel casting trays , which are available in a variety of sizes and composed of UV transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. Sample combs , around which molten medium is poured to form sample wells in the gel. Electrophoresis buffer , usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Loading buffer , which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.

12. Staining : DNA molecules are easily visualized under an ultraviolet lamp when electrophoresed in the presence of the extrinsic Fluor ethidium bromide. Ethidium bromide is a known mutagen or carcinogenic and should be handled as a hazardous chemical - wear gloves while handling To prepare gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, and heated in a microwave oven to melt it. Ethidium bromide is added to the gel (final concentration 0.5 ug/ml) to facilitate visualization of DNA after electrophoresis.

13. After cooling the solution to about 60℃ it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. Samples containing DNA mixed with loading buffer are then pipetted into the sample wells, the lid and power leads are placed on the apparatus and a current is applied run the gel for 30-45 mins at 100-150V. The current flow can be confirmed by observing bubbles coming off the electrodes DNA will migrate towards the positive electrode, which is usually colored red, in view of its negative charge and separated based on their isoelectric points.

14. Technique combining ideas of isoelectric points and electric fields. It gives good separation with a high resolution compared to any other method

15. CALCULATING pH After the time period of electrophoresis, the power is turned off and the gel is blot dried. The band containing protein sample is cut into pieces and the distance of the band from any one of the electrode is calculated. The band is then incubated in a solution of Potassium chloride, by doing this pH of the band is obtained. A graph is plotted taking pH of the band on X-Axis and distance of band on Y-Axis. A standard curve is obtained and pI value is calculated.

16. APPLICATIONS Widely used for separation and identification of serum proteins. Used in food and agricultural industries, forensic and human genetics laboratories. Used in enzymology, immunology and membrane biochemistry. 2D Gel electrophoresis is an application of IEF. Protein is first separated based on pI and then based on molecular weight using SDS-PAGE.