1. Electrophoresis / SDS-PAGE

2. Electrophoresis
The migration of proteins is affected by multiple factors involving their structural organization.
Amino acids can carry either a net positive, net negative, or neutral charge depending on the combination of amino acids they contain.
To make protein migration rates a function of molecular weight, it is necessary to impose a uniform shape and charge on all of the proteins in the mixture.

3. Why Use Polyacrylamide Gels to Separate Proteins?
Smaller pore size than agarose
Proteins much smaller than DNA
average protein = kD
“average” DNA = >2000 kD

4. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
CH2
CH3
SDS detergent
negative charge added to proteins
SDS

5. Why heat the samples?
Heating the samples helps denature proteins and protein complexes, allowing the separation of individual proteins by size
s-s - +
SDS, heat
proteins
with SDS

6. - How does SDS-PAGE work? +
Proteins (negatively charged due to SDS) move to positive electrode
Proteins separate by size
Smaller proteins move faster - +
largest
large
small
smallest