ISOELECTRIC FOCUSING

Isoelectric focusing, its history and how it is performed.

1. ISOELECTRIC FOCUSING PAUL SINGH

2. ISOELECTRIC FOCUSING Isoelectric 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 also known as electrofocusing ,

3. HISTORY

4. HISTORY IEF began in 1964, when Olaf Vesterberg filed a Swedish patent on the synthesis of new chemicals called carrier ampholytes . This technique was popularized by H.Svensson in Sweden. Highly efficient. If paper electrophoresis resolves plasma proteins into six bands, Isoelectric focussing resolves it into at least 40 bands.

5. CARRIER AMPHOLYTE

6. CARRIER 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 behave as a behave and offer conductance.

7. BASICS OF ISOELECTRIC FOCUSING

8. If the number of acidic groups in a protein exceeds the number of basic groups, the pI of that protein will be at a low pH value and the protein is classified as acidic. Similarly if the number of basic groups in a protein exceeds the number of acidic groups, the pI of that protein will be at a high pH an the protein is classified as basic. Proteins show a pI value of 4-7 with the pH falling in the range of 3-12.

9. 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.

10. PROCEDURE

11. PREPARATION OF GEL CHEMICALS REQUIRED :- ACRYLAMIDE, AMMONIUM PERSULPHATE(APS), TEMED, CARRIER AMPHOLYTE, UREA, WATER Required amount of acrylamide is dissolved in distilled water. Remove air bubbles if present. Add solution containing of carrier ampholytes to the mixture. Mix thoroughly Add 10%APS and TEMED Pour the mixture into the gel cassette. Place the comb in the gel cassette Allow the gel to solidify Remove comb after solidification.

12. SET UP OF THE APPARATUS Connect the terminals of the power supplier to electrodes of the gel chamber. (Black= Cathode ; Red= Anode) Pour catholyte (Sodium hydroxide) in the upper buffer chamber and Anolyte (Phosphoric acid) in the lower buffer chamber

13. SAMPLE PREPARATION AND LOADING Protein sample is mixed with a equal amount of gel loading buffer. Mix the sample and the buffer thoroughly. Load the sample onto the wells. After loading, run the gel for 30 mins at 150V and for 2 hours at 200V. Samples are separated based on their isoelectric points.

14. 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.

15. FIXING AND STAINING OF GEL Soak the gel in 10%TCA for removal of ampholytes Ampholytes are removed to avoid background staining Stain the gel using coomasie blue for 10min. Discard staining solution and place the gel in destaining solution.

16. APPLICATIONS

17. 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.

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