SDS-PAGE.pptx

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1. Sodium Dodecyl Sulphate (SDS) M.Phool Badshah

2. Principal: SDS (also called sodium lauryl sulphate ) is an anionic detergent meaning that when dissolved its molecules have a net negative charge within a wide PH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and strongly attracted toward an anode (positive charged electrode) in an electric field. SDS-PAGE

3. Poly acrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured the polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. aa

4. Conc. of resolving and stacking gel used: Re a Resolving Gel ( 7.5 ml ) Water ( 2.475ml ) Acrylamide ( 3ml ) 1.5 M Tris HCL ( 1.875ml ) 10% SDS ( 75micro-liter ) 10% APS ( 75microliter ) TEMED ( 3micro-liter ) Stacking Gel ( 2.5ml ) Water ( 1.720ml ) Acrylamide ( 495 microliter ) 1M Tris HCL ( 312.5microliter ) 10% SDS ( 25micro-liter ) 10% APS ( 25 micro-liter ) TEMED ( 3micro-liter )

5. Wash glass plates and spacers in warm detergent solution and rinse with tap water deionized water. Rinse plates with ethanol and dry it. The glass plates musd be free of grease spots to prevent air bubbles in gel. Assemble the glass plates with spacers. Prepare Gel solution with desired poly acrylamide percentage, which gives amount of each component required to make 100ml. Add 35 micro-liter of TEMED for each 100ml of acrylamide , bis solution, and mix the solution with gentle swirling. Gels can be cast with 1 micro-liter TEMED per ml of gel solution to increase rate of polymerization. Immediately insert comb into gel, being careful not to allow air bubbles become trapped under teeth. Allow acrylamide to polymerize for 30-60 minutes at room temperature. How to run SDS ?

6. After polymerization is complete, surround comb and top of gel with paper towels soaked in 1X TBE. Seal the entire gel and store it at 4’C untill needed. When ready to proceed for electrophoresis Squirt 1X TBE buffer, pull comb form polymerized gel. Use syringe to rinse out wells with 1X TBE, remove tape. Attach gel to electrophoresis tank, using clips on sides. Fill reservoirs of electrophoresis tank with TBE buffer. Use syringe to flush out wells once more with 1X TBE. Mix the DNA samples with appropriate amount of 6X-Gel loading buffer. Load the mixture into wells using micro-pipette. Connect electrodes with power pack (positive electrode connect to bottom reservoir), turn on the power and begin the electrophoresis run. aa

7. Run the gel untill the marker dyes have migrated the desired distance. Turn off electric power, disconnect the leads, and discard the electrophoresis buffer from the reservoirs. Detect the positions of bands of DNA in the polyacrylamide gel. a

8. Page has high loading capacity, up to 10 micrograms of DNA can be loaded into single well without significant loss of resolution. Page is an ideal system from which isolate DNA fragments for sub-cloning and other molecular biological techniques. About PAGE :

9. Separate from other proteins on the basis of molecular weight and size. Determine molecular size of protein. Determine quantifies amount present. Used in western blot assay. Uses of PAGE: