SDS-PAGE electrophoresis is a technique used to separate proteins by size. It involves running proteins through a stacking gel and resolving gel with an electric current. The stacking gel concentrates the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences. Key components of SDS-PAGE include SDS to impart identical charge-to-mass ratios to proteins, reducing agents to unfold proteins, and polyacrylamide gels which sieve proteins during electrophoresis based on their size.

1. SDS-PAGE Electrophoresis
Excellent resources on the web:
Wikipedia
mullinslab.ucsf.edu/protocols/html/SDS_PAGE_protocol.htm
Good videos, e.g., one by UC Davis people, available
on YouTube:
http://sdspage.homestead.com/
http://www.molecularstation.com/wiki/SDS-PAGE_protocol

2. An overview of SDS-PAGE electrophoresis
Stacking gel,
pH 6.8
Resolving gel
pH 8.8
anode cathode
Details will be discussed after the overview

3. Some of the chemical components used

4. A typical, stained SDS-PAGE gel

5. What do you notice
about the ordinate
axis?

6. This figure will look familiar when
you start next week’s THQ

7. What you will be doing
for next week’s take-home
quiz (and for
next week’s lab)

8. Isoelectric focusing

12. Resolution of 1000 different proteins from E. coli

13. Electrophoresis – some theory
It involves the movement of charged particles under
the influence of an electric field
Very useful for separating amino acids, proteins,
oligonucleotides, DNA, RNA, and DNA sequencing
Negatively charged molecules move towards the
positive (+) electrode (the anode in this set-up)
Positively charged molecules move towards the
negative (-) electrode (the cathode in this set-up)

14. Theory – cont’d
Remember from General Chemistry:
cathode is where reduction occurs (consonants)
anode is where oxidation occurs (vowels)
In an electrolytic cell, the cathode is negatively
charged and can “give electrons away” (reduction);
the anode is positively charged and can “take
electrons away” (oxidation).
In a battery, the convention is reversed: the anode
has a negative charge and the cathode has a positive
charge.

15. Electrode Reactions
Cathode (reduction): 2e- + 2H2O Û 2OH- + H2­
Anode (oxidation): H2O Û 2H+ + ½O2­ + 2e-
Twice as many bubbles arise from the cathode. pH
increases in the cathode chamber. pH decreases in
the anode chamber.

16. Theory cont’d
From electrostatics:
where q is the charge of the particle, E is the electric
field strength, V is the electric field potential in volts,
and d is the distance between electrodes.

17. Theory cont’d
Also, the movement of the charged particle in the
electric field is opposed by a frictional force:
where r is the Stoke’s (effective) radius of the
particle, h is the viscosity, v is the velocity, and f
is the frictional coefficient (6prh)

18. Theory cont’d
In a constant electric field (constant velocity) the
two forces balance (no acceleration) and we have:
And we define the mobility (m) as follows:

19. Theory cont’d
Key idea: mobility is directly proportional to the
charge of the molecule and inversely proportional to
the frictional coefficient, which itself depends on the
size and shape of the molecule.
the relative mobilities of different molecules
depends on charge, size, and shape (all three factors)

20. If electrophoresis occurred in a non-viscous solution,
local heating effects would generate convective flows,
which would interfere with the orderly separation of
molecules. A stabilizing medium (i.e., gel) is used to
greatly improve separation.
Gels are a cross between a solid and a liquid;
mechanically more stable than a liquid but they are
not solid. Liquids are retained, and small molecules
can pass through relatively freely.
In SDS-PAGE, the polyacrylamide is cross-linked
by N,N¢-methylene-bis-acrylamide, which provides
a molecular sieving effect.

21. acrylamide, a nerve
toxin! (Gloves)
TEMED, helps in cleavage
of persulfate
persulfate
homolytic
cleavage
·
free radical

22. Free radical attacks acrylamide (steals an electron)
and in the process forms a cation radical + sulfate ion
·
Cation radical attacks other monomers to form a
polymer

23. So you end up with
a polymer of
acrylamide, with
occasional methylene-bisacrylamide
cross-links

24. What is the purpose of using SDS?
SDS binds to denatured proteins in
a ratio of 1.4 g SDS/g protein
Since proteins are denatured, their shapes
are similar and the high density of negative
charges due to the SDS impart
approximately the same charge-to-mass
ratio for all proteins. Thus, separation is
effected by differences in size.
Reducing agents (e.g., b-mercaptoethanol)
to reduce disulfide bonds

25. An overview of SDS-PAGE electrophoresis
Stacking gel,
pH 6.8
Resolving gel
pH 8.8
anode cathode

26. Why does the stacking gel have a lower
concentration of acrylamide and why is it at a
different pH than that of the resolving gel?
Under the influence of the electric field, the Cl- ions
(from the Tris×Cl) move relatively fast through the
stacking gel towards the anode due to their small size.
Many of the glycine molecules in the running buffer
(pH 8.3) are initially attracted toward the anode, but
when they encounter the stacking gel at pH 6.8, they
become protonated (and therefore zwitterionic) and
mostly neutral and relatively immobile. What fraction
is in the glycinate form at this pH?

27. So, in the stacking gel the Cl- ions are moving ahead
toward the anode, and the glycine molecules, which
are mostly neutral, are lagging behind and not
carrying the charge. The fast moving Cl- ions and
slow, mostly neutral glyicine molecules create a zone
of low conductance or high resistance. Due to the
requirement of constant current (I) in an electrical
circuit (E = IR), the high resistance leads to a localized
(narrow zone) increase in the electric field. The
negatively charged protein molecules find themselves
in between the Cl- ions and the glycine molecules and
thus subject to the high field strength. They move
forward in the field fairly rapidly, until they encounter
the interface of the stacking gel and the resolving gel.

28. The key is that the proteins cannot move past the
Cl- ions. To do so would put them in a region of high
conductance and low field strength, which would
cause them to slow down.
Thus, the proteins end up moving through the
relatively porous (low concentration of acrylamide)
stacking gel (sieving effect not desirable here) in the
moving zone of high voltage (V = IR) and they reach
the “starting gate” (the interface between the stacking
and resolving gels) in a very thin band and at
essentially the same time.

29. The resolving gel has a higher pH (8.8), which leads
to a higher proportion of glycine molecules becoming
deprotonated and thus negatively charged. These
glycinate ions overtake the proteins due to their
small size. The sieving characteristics of the gel
can now separate proteins on the basis of size.

30. But the need to pour stacking gels may be obviated: