Isoelectric Focusing.pptx

Macro molecule separation technique in biochemistry. PAGE; Isoelectric Focusing. DNA AND RNA separation techniques. nucleotide separation techniques.

1. Polyacrylamide gel electrophoresis and IEF PRESENTED BY: FARWA MUKHTAR PRESENTED TO:MAM ZARAFSHAN COURSE TITLE:STRUCTURE AND FUNCTION OF MACROMOLECULES COURSE CODE:ZOL-501

2. Introduction: PAGE is a technique widely used in life science laboraties to separate biological macromolecules usually protein or nucleic acid according to their electrophoretic mobility. PAGE is a standard method used to separate,identify and purify biopolymers,since porous in nature. The most commonly used form of PAGE is SDS-PAGE used mostly for separation of protiens .

3. Principle: SDS-PAGE is the analytical principle used for separation of proteins depend upon size. The technique is based upon the principle that a charged molecule will migrate in an electric field towards an electrode with opposite sign. The general electrophoresis techniques cannot be used to determine the molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size.

4. Conti… To overcome this, the biological samples needs to be treated so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. For this different protein molecules with different shapes and sizes, needs to be denatured so that the proteins lose their secondary, tertiary or quaternary structure. The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode are separated by a molecular sieving effect based on size. After the visualization by a staining technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder.

5. Requirements Acrylamide solutions (for resolving & stacking gels). Isopropanol / distilled water. Gel loading buffer. Running buffer. Staining, destaining solutions. Protein samples Molecular weight markers. The equipment and supplies necessary for conducting SDS-PAGE includes: An electrophoresis chamber and power supply,Glass plates (a short and a top plate) , Casting frame,Casting stand,Combs .

6. Steps: Sample preparation: Samples may be proteins or nucleic acids. The sample mixed with a chemical denaturant, usually SDS for proteins or urea for nucleic acids. SDS is an anionic detergent that denatures secondary and non–disulfide–linked tertiary structures. Applies a negative charge to each protein in proportion to it’s mass.

7. Continue Heating the sample at 60°C further promotes denaturation. A tracking dye used to track the progress of solution through gel during electrophoretic run.

8. Preparation of polyacrylamide gel: Gel consist of acrylamide,bisacrylamide,SDS or urea and buffer. Ratio of bisacrylamide to acrylamide can be varied for special purposes. Concentration of acrylamide gel can also be varied,generally in range from 5% to 25%. Lower percentages are better than higher percentages of gel. Gel is polymerized between two glass plates in glass caster with comb.

9. After the gel is polymerized, comb is removed and gel is ready for electrophoresis.

10. Electrophoresis: Buffer used at anode & cathode may be same or different. Electric field is applied across the gel,causing negatively charged proteins to migrate away from negative & towards positive electrode(anode). Depending on size,each biomolecule move differently through gel matrix. Small molecules easily move through pores of gel than larger ones. Gel is run for few hours. After set amount of time,the biomolecules will have migrated different distances.

11. Continue Smaller molecules travel farther down the gel while larger ones remain closer to point of origin. Biomolecules separated roughly depend mainly on molecular weight.

12. Conti… Detection: Following electrophoresis,gel may be stained. For protein with autoradiography,for nucleic acid or for either silver stain. After staining,different biomoleculed appear as distinct bands within gel.

13. Goals: Separate mixed sample of proteins to identify and qualify single protein from mixture. Starting sample comes from number of sources such as patient sample or bacterial culture. It is also used to separate DNA and RNA. But proteins are most most common sample type PAGE is often combined with a technique called western blotting. Blotting uses antibodies to bind to specific protein antigens. And allow us to identify individual protein with high specificity.

14. Applications: Measuring molecular weight. Peptide mapping. Estimation of protein size. Determination of protein subunits or aggregation structures. Estimation of protein purity. Protein quantitation. Monitoring protein integrity.

15. Advantages: Stable chemically cross-linked gel Greater resolving power (Sharp bands) Can accommodate larger quantities of DNA without significant loss in resolution The DNA recovered from polyacrylamide gels is extremely pure The pore size of the polyacrylamide gels can be altered in an easy and controllable fashion by changing the concentrations of the two monomers. Good for separation of low molecular weight fragments

16. Disadvantages: Generally more difficult to prepare and handle, involving a longer time for preparation. Toxic monomers Gels are tedious to prepare and often leak Need new gel for each experiment Stable chemically cross-linked gel.

17. Isoelectric focusing: IEF is a method of separating proteins according to their pI in a pH gradient. Isoelectric point is denoted as pI . pI is defined as the pH at which protein carry no net charge. Or the pH at which protein become immbolize in an electric field.

18. Diagram:

19. Principle: Protein separation/purification can be easily done if pI of protein is known. The resolution of isoelectric focusing is very high. In normal electrophoretic method,pH between anode & cathode remains constant. But in IEF,pH gradient is arranged when pH of protein below it’s pI proteins become positively charged. And it will migrate towards cathode because of the pH gradient charge of protein molecule changes.

20. Conti… While moving forward,there will be a point at which net charge of protein become zero this point is called isoelectric point. When a single or mixture of protein is run under an electric field at specific pH the protein stops moving .

21. Diagram:

22. Procedure: Preparion of IEF gel Addition of Ampholytes Generation of pH gradient Staining

23. Staining: Staining is done by Coamassie brilliant blue stain. But it can not be done directly because Ampholytes will stain too,directly given the blue gel. Gel is first washed with fixing solution(10% trichloro acetic acid). This precipitates protein of the gel. It allow much smaller Ampholytes to be washed out. After that,gel is stained with Coamassie brilliant blue and then destained .

24. Advantages: Efficient Easy( clear,one dimensional separation of protein). Fast Economic(no sophistricated equipments required). Disadvanges : The disadvange of IEF is that minor bands and aging bands are also seen It cause confusion in interpretion .