SDS­PAGE

Sodium Dodecyl Sulfate ­  Polyacrylamid Gel  Electrophoresis

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

Sodium Dodecyl Sulfate

SDS

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
q q

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 q SDS nonpolar chains arrange themselves on proteins and
destroy secondary tertiary and quarternary structrure q Thus shape is no longer an issue as the protein SDS complex becomes rod shaped

q In aqueous solutions, SDS polarizes releasing Na+ and retaining a negative charge on the sulfate head q So much SDS binds to proteins that the negative charge on the SDS drowns out any net charge on protein side chains q In the presence of SDS all proteins have uniform shape and charge per unit length

Polyacrylamide Gels
q

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

NH2 CH2 NH2
Acrylamide

CH2 CH

bis-Acrylamide

Polyacrylamide Gels
q

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

O C CH2 CH NH2

O C CH2 CH SO4-. NH2

Polyacrylamide Gels
q

Acrylamide polymerizes in the presence of free radicals typically supplied by ammonium persulfate q TMED (N,N,N’,N’-tetramethylethylenediamine) serves as a catalyst in the reaction
O C CH2 CH NH2 O C NH2 O C NH2 O C NH2

CH2 CH

CH2 CH

CH2 CH

SO4-.

q

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

Polyacrylamide Gels

CH2 CH

CH2 CH

CH2 CH

CH2 CH

CH2 CH

bis-Acrylamide

q

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

Polyacrylamide Gels

q

Pore size in gels can be varied by varying the ratio of acrylamide to bis-acrylamide q Protein separations typically use a 29:1 or 37.5:1 acrylamide to bis ratio

Polyacrylamide Gels

Littleof bis-acrylamide Lots bis-acrylamide

SDS­PAGE
1 2 3

3 1
Addition of SDS 2 Protein becomes rodshaped with uniform charge distribution