1. SDS-PAGESodium Dodecyl Sulfate - Polyacrylamid Gel Electrophoresis

2. Sodium Dodecyl Sulfate • SDS is a common ingredient in detergents • Other names for SDS include laurel sulfate and sodium laurel sulfate • As a detergent SDS destroys protein secondary, tertiary and quaternary structure • This makes proteins rod shaped • SDS also sticks to proteins in a ratio of approximately 1.4 g of SDS for each gram of protein • Negative charge on the sulfate groups of SDS mask any charge on the protein

3. O H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+ O Non-polar Hydrophobic tail Polar Hydrophilic head H H H H H H H H H H H H H H H H H H H H H H H H SDSSodium Dodecyl Sulfate C12H25NaO4S • Because it is amphipathic, SDS is a potent detergent

4. SDS Protein SDS and Proteins

5. SDS and Proteins • SDS nonpolar chains arrange themselves on proteins and destroy secondary tertiary and quarternary structrure • Thus shape is no longer an issue as the protein SDS complex becomes rod shaped • In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head • So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains • In the presence of SDS all proteins have uniform shape and charge per unit length

6. O Acrylamide C NH2 CH2 CH CH2 CH O C NH2 CH2 Acrylamide Acrylamide O C NH2 bis-Acrylamide CH2 CH Polyacrylamide Gels • Polyacrilamide is a polymer made of acrylamide (C3H5NO) and bis-acrilamide (N,N’-methylene-bis-acrylamide C7H10N2O2)

7. O O C C NH2 NH2 CH2 CH2 CH CH SO4-. Polyacrylamide Gels • Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate

8. O O O O C NH2 C NH2 C NH2 C NH2 CH2 CH CH2 CH CH2 CH CH2 CH SO4-. Polyacrylamide Gels 1. Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate • 2.TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction

9. O O O C NH2 C NH2 C NH2 CH2 CH CH2 CH CH2 CH CH2 CH O O O C NH2 C NH2 C NH2 CH2 O CH2 CH CH2 CH O C NH2 C NH2 CH2 CH CH2 CH bis-Acrylamide Polyacrylamide Gels • bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

10. Polyacrylamide Gels • bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

11. Lots of bis-acrylamide Little bis-acrylamide Polyacrylamide Gels • Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio • Pore size in gels can be varied by varying the ratio of acrylamide to bis-acrylamide

12. 3 Addition of SDS 2 Protein becomes rod-shaped with uniform charge distribution 1 SDS-PAGE 1 2 3

13. The End