SDS- PAGE METHOD

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) method is one of the
most commonly methods used to determine the molecular weight of an unknown protein because
it doesnt require using large amounts of highly purified proteins and costly equipment.
Unlike nucleic acids, proteins have complex shapes and variety of charges. SDS is a detergent
that has an anionic head and a lipophilic tail. SDS binds to protein with a stoichiometry of one
SDS molecule per two amino acids to denature the protein. SDS-PAGE (Sodium dodecyl sulfate
polyacrylamide gel electrophoresis) is a technique based on the migration of the proteins by the
pore size of the gel matrix and charge, size, and shape of protein to create relative charge
corresponding the protein mass in order to determine molecular weight of protein or to check
the purity of protein. SDS-PAGE is used to determine the most abundant proteins from
a complex sample and compare different production batches on a single gel.
The system actually consists of two gels
- a resolving (aka running) gel in which proteins are resolved on the basis of their
molecular weights (MWs)
- a stacking gel in which proteins are concentrated prior to entering the resolving gel.
Dierences in the compositions of the stacking gel and resolving gel and electrophoresis
buer produce a system that is capable of finely resolving proteins according to their
MWs.
Smaller proteins diffused rather quickly out of the gel, while the larger ones at
the top diffused much more slowly, and took the stain. The acidified alcohol in
the stain solution and in the destain solutions is essential, as it precipates
proteins, preventing them from diffusing out of the acrylamide matrix
- Purpose of the stacking gel: to concentrate all the proteins in the sample into a thin band
at the top of the resolving gel. Makes it possible to use a dilute sample
- Purpose of the resolving gel: to separate the proteins based on size. Two gel layers with
different polyacrylamide concentrations. A different buffer for each of the two parts of
the gel. And a third buffer as the running buffer.
- Stacking gel: It has large pores, so proteins of all sizes move easily through the pores of
the stacking gel until they meet a frictional barrier at the top of the resolving gel, with its
smaller pores.
- Using the stacking and running buffers to form a voltage gradient => increase protein
mobility
Stacking gel buffer is low salt concentration and Cl- is leading ion because it is
small, negatively charged, and moves quickly through gel
Running buffer ion is primarily glycine = trailing ion, which at pH 6.8 is
nearly neutral
 A region of low ionic strength quickly develops between Cl- and glycine,
generating a voltage gradient. Large, negatively charged proteins are left to
constitute most of the molecular current, and move quickly to the bottom of the
stacking gel. The resolving gel separates proteins as a function of percentage
acrylamide, ratio of acrylamide to bis, extent of difference in size between the
proteins being resolved
The resolving gel has a higher [ion] (0.75 M Tris-H+Cl-) than the stacking gel
(0.25 M Tris-H+Cl-) so proteins contribute less to the total ionic current than
they did in the stacking gel, and as a result the mobilities of proteins reduce and
differences in mobilities among proteins of different sizes become more
apparent.
In addition, the resolving gel has a higher pH (8.8) than the stacking gel (6.8) so
glycine takes on a more negative charge, thereby the increasing the total ion
concentration and because it is small, moves ahead of the proteins. So proteins
move slowly through the gel and are resolved by friction on the basis of size
- We add TEMED and APS at last steps because the solution will quickly polymerization
when we add TEMED and APS. So that prevent gel from forming before we pour it in
SDS-PAGE system
 A
The larger proteins that are filtered from gel filtration do not make up the matrix on the gel
plate and they form the void volume of the column. In this case, the planes 21-26 contain
proteins that eluted earlier and run at higher molecular weight on the gel than the proteins in
lanes 27-36. Moreover, in the fraction from 21 to 26 there was one band in each column and
this make the void space below, whereas, there were many bands in the column from 27 to
36. Therefore, we can conclude that the protein in fractions 2126 should be bigger than the
protein(s) in fractions 2736.