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

4. SDS and Proteins
SDS
Protein

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

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

8. 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
NH2 O CH
CH2 C O CH
CH2
NH2 C
SO4-.

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

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

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

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

13. The
End