Sds page (sds-polyacrylamide gel electrophoresis- )

I AM HAFIZ MUHAMMAD WASEEM from mailsi vehari
BSc from science college Multan
MSC university of education Lahore
i love Pakistan and my teachers

1. HAFIZ MUHAMMAD WASEEM BA BA G LAHORE Micrometry

2. SDS- Polyacrylamide Gel Electrophoresis- Introduction Advanced Analytical Techniques UNIVESITY OF EDUCATION LAHORE PAKISTAN

3. Introduction What is SDS-PAGE? Separation of macromolecules on the basis of their electro- phoretic mobility is called electrophoresis The techniques cannot be used to determine molecular weight of biological molecules because the mobility of a substance in the gel depends on both charge and size So, there is a need to denature the proteins with some detergent like SDS so that they lose their secondary, tertiary or quaternary structures and become linear  

4. Introduction This method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)  The method is also called Laemmli method after Laemmli who was the first to publish a paper employing SDS-PAGE A polypeptide chain binds with SDS in proportion to its relative molecular mass This results in fractionation by approximate size during electrophoresis

5. Introduction Negative charges on SDS destroy most of the complex structure of proteins These molecules are strongly attracted toward an anode in an electric field Polyacrylamide gels restrain larger molecules from migrating as fast as the smaller molecules SDS-PAGE is the most widely used analytical method in biochemistry and molecular biology Characterization of proteins is also possible

6. Advanced Analytical Techniques SDS- Polyacrylamide Gel Electrophoresis - Principle

7. Principle of SDS-PAGE SDS denatures the proteins by binding to hydrophobic regions Non-covalent bonds are disrupted and the proteins acquire a net negative charge A Concurrent treatment with a disulfide reducing agent such as β- mercaptoethanol or DTT ( dithiothreitol ) also denature the proteins by reducing disulfide linkages Proteins samples get uniform structure and charge A suitabe dye is used to monitor electrophoresis

8. Separation depends on their molecular weight only Small proteins migrate faster than the larger ones through the gel matrix under the influence of the applied electric field Number of SDS molecules that bind is proportional to the size of the protein So, the SDS-treated proteins get similar charge-to-mass ratios and similar shapes move towards anode and separate only according to their molecular weight Principle of SDS-PAGE

9. SDS- Polyacrylamide Gel Electrophoresis - Procedure Advanced Analytical Techniques

10. Sample containing proteins or nucleic acids is prepared by using Homogenizer Sonicator   Filtration & centrifugation It is mixed with some suitable denaturant like SDS for proteins Proteins are also heated with a reducing agent like Beta- mercaptoethanol which denatures the proteins by reducing disulfide linkages thus breaking the complex structures Procedure of SDS-PAGE Sample Preparation

11. SDS-PAGE gels consist of separating and stacking gels For separating gel prepare gel solution (desired %age) and pour it into the gap between the glass plates It is degassed under a vacuum or butanol is added to prevent the formation of air bubbles during polymerization  APS and TEMED are added to initiate polymerization Wash top of the gel for several times to remove acrylamide that is unpolymerized Procedure of SDS-PAGE Preparing the gel

12. Stacking gel (5%) is poured on top of the separating gel and a comb is inserted to make wells After polymerization the comb is removed AZis ready for electrophoresis Acrylamide concentration may range from 5% to 25% Lower percentage gels are better for resolving very high molecular weight molecules While higher percentages are needed to resolve smaller proteins Finally the gel is fixed in the electrophoresis chamber Procedure of SDS-PAGE Preparing the gel

13. Samples are mixed with loading buffer and are loaded in the wells The ladder is loaded in the first well Various buffer systems are used in PAGE depending on the nature of the samples Generally the gel is run for 1 hour at 120V voltage for 12% separating gel Negatively charged proteins or nucleic acids migrate towards the anode Smaller molecules move faster than the larger ones Procedure of SDS-PAGE Electrophoresis

14. After electrophoresis, the gel is stained with dyes like Coomassie Blue Different Proteins are stained as distinct bands in the gel Different proteins are stained differently with the stains Detergents like SDS are used to separate the folded proteins A suitable marker is used to estimate molecular mass of the unknown proteins Procedure of SDS-PAGE Staining & Visualization

15. SDS- Polyacrylamide Gel Electrophoresis - Factors Advanced Analytical Techniques

16. A number of factors affect the rate of electrophoresis like Concentration of gels Size of molecules being electrophoresed Voltage used Time Ionic strength of buffers Dyes such as ethidium bromide used during electrophoresis Characterization of proteins by Western blotting by transferring to a membrane Factors affecting SDS-P AGE

17. SDS- Polyacrylamide Gel Electrophoresis - Applications Advanced Analytical Techniques

18. SDS-PAGE has a number of applications which include Measuring molecular weights of different bio-molecules Estimation of purity of the proteins Quantification of proteins Analysis of number and size of proteins subunits Characterization of proteins by Western blotting Staining of proteins Labeling of different proteins Protein mapping Applications of SDS-PAGE