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 A technique widely used to separate

proteins according to their electrophoretic

 SDS gel electrophoresis of samples having

identical charge per unit mass due to
binding of SDS results in fractionation by
size and is probably the world's most widely
used biochemical method
 C12H25NaO4S
 MW: 288.38

 most common dissociating agent used to

denature native proteins to
individual polypeptides

 When a protein mixture is heated to 100 °C

in presence of SDS, the detergent wraps
around the polypeptide backbone. It binds
to polypeptides in a constant weight ratio of
1.4 g/g of polypeptide
 SDS is a negatively charged detergent.
 Disrupts secondary and tertiary protein structures
by breaking hydrogen bonds and unfolding protein.
 ‘Masks’ charge on protein so that all proteins act
the same as regards charge.
 Prevents protein aggregation.
 Prevents protein shape from influencing gel run.
 Samples may be taken from whole tissue or
cell culture
 Combination of biochemical and mechanical
techniques can be used to separate different
cell compartments and organelles
 The solution of proteins to be analyzed is
mixed with SDS, an
anionic detergent which denatures secondary
and non–disulfide–linked tertiary structures,
and applies a negative charge to each protein
in proportion to its mass
 A tracking dye may be added to the protein
solution (of a size smaller than protein) to
allow the experimenter to track the
progress of the protein solution through the
gel during the electrophoretic run
 the gel may be stained allowing
visualization of the separated proteins, or
processed further
 different proteins will appear as distinct

bands within the gel. It is common to

run molecular markersof known molecular
weight in a separate lane in the gel, in order
to calibrate the gel and determine the
weight of unknown proteins by comparing
the distance traveled relative to the marker
 The gel is actually formed because the
acrylamide solution contains a small amount.
 Gel electrophoresis is usually the first choice
as an assay of protein purity due to its
reliability and ease. The presence of SDS and
the denaturing step causes proteins to be
separated solely based on size. False
negatives and positives are possible. A
comigrating contaminant can appear as the
same band as the desired protein. 
Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis

 A procedure to separate proteins and

determine their Molecular Weights.

 It follows the principle that a charged

molecule will migrate in an electric field
toward an electrode of opposite sign.
Electrophoresis in Principle:

Separation of charged molecules in electric field is

a function of:
• Relative mobility of charged species (related to
frictional resistance which is related to size).
• Charge on the species.
• Will migrate toward cathode (-) or anode (+).
• Separation occurs due to different rates of
migration due to magnitude of charge and
frictional resistance (related to size).

Rf = (Z E)
• Z = charge on molecule
• E = Voltage applied (driving force)
• f = frictional resistance

Rf is measured by:
Rf = Distance protein band moves
Distance dye front moves
 Extract Protein
 Solubilize and Denature Protein
 Separate Proteins on a gel
 Stain proteins (visualization)
 Analyze and interpret results
Primary structure = linear chain of
amino acids

• Secondary structure = domains of

repeating structures, such as β-pleated
sheets and α-helices

• Tertiary structure = 3-dimensional

shape of a folded polypeptide,
maintained by disulfide bonds,
electrostatic interactions, hydrophobic

• Quaternary structure = several

polypeptide chains associated together
to form a functional protein

•SDS (Sodium Dodecyl CH2

Sulfate) detergent CH2
–solubilizes and CH2

denatures proteins CH2

–negative charge to CH2

proteins CH2

•Heat denatures proteins



 The amino acid cysteine contains a
sulfhydryl (-SH) group that spontaneously
forms a disulfide bond (-S-S-) with another
sulfhydryl group under normal intracellular
conditions. Disulfide bonding is covalent
and is not disrupted by SDS.

 DTT is a strong reducing agent. Its specific

role in sample denaturation is to remove
the last bit of tertiary and quaternary
structure by reducing disulfide bonds.
•Negatively charged
proteins move to
positive electrode s-s

SDS, heat

•Smaller proteins proteins with

move faster

• Proteins separate -
by size

 Size measured in daltons (Da) or kilodaltons (kDa)
 Dalton = atomic mass unit
= corresponds to mass of hydrogen
molecule (1.66 x 10 -24 gram)
= defined also as 1/16 of the mass of an
atom of oxygen
 Average amino acid = 110 Da
Average nucleotide pair = 649 Da
 Proteins
 Muscles contain many proteins, some of which are
variable from organism to organism
 Actin and Myosin
◦ Form muscle fibers that allow muscles to contract and
◦ Most common muscle proteins in all animals
Lane 1. Kaleidoscope Markers
2. Shark
3. Salmon
4. Trout
5. Catfish
6. Sturgeon
7. Actin and Myosin Standard
Kaleidoscope standard
Myosin – blue
β -galactosidase – magenta
Bovine serum albumin – green
Carbonic anhydrase – violet
Soybean trypsin inhibitor – orange
Lysozyme – red
Aprotinin - blue
Protein kDa Function
titin 3000 center myosin in sarcomere
dystrophin 400 anchoring to plasma
filamin 270 cross-link filaments into gel
myosin heavy chain 210 slide filaments
spectrin 265 attach filaments to plasma
nebulin 107 regulate actin assembly
α -actinin 100 bundle filaments
gelosin 90 fragment filaments
fimbrin 68 bundle filaments
actin 42 form filaments
tropomyosin 35 strengthen filaments
myosin light chain 27 slide filaments
troponin (T, I, C) 30, 19, 17 mediate regulation of
thymosin 5 sequester actin
• Pairwise comparisons between species

# of proteins in common
Total # of unique proteins
Protein Color MW (daltons) on Tris
HCl gel

Myosin Blue 204,649

B-galactosidase Magenta 127,511

Bovine serum Green 85,130


Carbonic anhydrase Violet 37,830

Soybean trypsin Orange 30,906


Lysozyme Red 27,230

Aprotinin Blue 6,638