Sds-page

SDS­PAGE Sodium Dodecyl Sulfate ­  Polyacrylamid Gel  Electrophoresis

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 ●

SDS

Sodium Dodecyl Sulfate

C12H25NaO4S

H H H H H H H H H H H H O H-C-C-C-C-C-C-C-C-C-C-C-C-O-S-O-Na+ H H H H H H H H H H H H O Non-polar Hydrophobic tail ● ●

Polar Hydrophilic head

Because it is amphipathic, SDS is a potent detergent SDS may also be called sodium lauryl sulfate and is a common ingredient of shampoos laundry detergent and other cleaning products

SDS and Proteins SDS

Protein

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

Polyacrylamide Gels ●

Polyacrilamide is a polymer made of acrylamide (C3H5NO) and bis-acrilamide (N,N’-methylenebis-acrylamide C7H10N2O2) CH2 CH

O C

O

C

NH2

CH2 CH Acrylamide

Acrylamide

NH2 CH2

O

C

CH2 CH

NH2 Acrylamide

bis-Acrylamide

Polyacrylamide Gels ●

Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate

O C CH2 CH

O NH2

C CH2 CH

SO4-.

NH2

Polyacrylamide Gels Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate ● TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction

●

O C CH2 CH

O NH2

C

CH2 CH

SO4-.

O NH2

C

CH2 CH

O NH2

C

CH2 CH

NH2

●

Polyacrylamide Gels

bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains O C CH2 CH

O NH2

C

CH2 CH O

O

C

C

CH2 CH

CH2 CH

O NH2

C

NH2

CH2 CH

NH2 CH2

O

NH2

C

CH2 CH

bis-Acrylamide

O NH2

C

CH2 CH

O NH2

C

CH2 CH

NH2

●

Polyacrylamide Gels

bis-Acrylamide polymerizes along with acrylamide forming cross-links between acrylamide chains

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

●

Little Lots ofbis-acrylamide bis-acrylamide

SDS­PAGE 1

2

3

3 1

Addition 2 of SDS Protein becomes rodshaped with uniform charge distribution