SDS-PAGE Electrophoresis

mullinslab.ucsf.edu/protocols/html/SDS_PAGE_protocol.htm

Excellent resources on the web:

Wikipedia

Good videos, e.g., one by UC Davis people, availableon YouTube:

http://www.molecularstation.com/wiki/SDS-PAGE_protocol

http://sdspage.homestead.com/

An overview of SDS-PAGE electrophoresis

Details will be discussed after the overview

Stacking gel, pH 6.8

Resolving gel pH 8.8

anodecathode

Some of the chemical components used

A typical, stained SDS-PAGE gel

What do you notice about the ordinateaxis?

This figure will look familiar whenyou start next week’s THQ

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

Isoelectric focusing

Resolution of 1000 different proteins from E. coli

Electrophoresis – some theory

It involves the movement of charged particles underthe influence of an electric field

Very useful for separating amino acids, proteins,oligonucleotides, DNA, RNA, and DNA sequencing

Negatively charged molecules move towards thepositive (+) electrode (the anode in this set-up)

Positively charged molecules move towards the negative (−) electrode (the cathode in this set-up)

Theory – cont’dRemember from General Chemistry:

cathode is where reduction occurs (consonants)

anode is where oxidation occurs (vowels)

In an electrolytic cell, the cathode is negativelycharged 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.

Electrode Reactions

Cathode (reduction): 2e− + 2H2O ⇔ 2OH− + H2↑

Anode (oxidation): H2O ⇔ 2H+ + ½O2↑ + 2e−

Twice as many bubbles arise from the cathode. pHincreases in the cathode chamber. pH decreases inthe anode chamber.

Theory cont’d

From electrostatics:

where q is the charge of the particle, E is the electricfield strength, V is the electric field potential in volts,and d is the distance between electrodes.

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, η is the viscosity, v is the velocity, and f is the frictional coefficient (6πrη)

Theory cont’d

In a constant electric field (constant velocity) thetwo forces balance (no acceleration) and we have:

And we define the mobility (μ) as follows:

Theory cont’d

Key idea: mobility is directly proportional to the charge of the molecule and inversely proportional tothe frictional coefficient, which itself depends on thesize and shape of the molecule.

∴ the relative mobilities of different molecules depends on charge, size, and shape (all three factors)

Theory cont’d

If electrophoresis occurred in a non-viscous solution,local heating effects would generate convective flows,which would interfere with the orderly separation ofmolecules. A stabilizing medium (i.e., gel) is used togreatly 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 moleculescan pass through relatively freely.

In SDS-PAGE, the polyacrylamide is cross-linked by N,N′-methylene-bis-acrylamide, which providesa molecular sieving effect.

acrylamide, a nervetoxin! (Gloves)

TEMED, helps in cleavageof persulfate

persulfate

homolyticcleavage

free radical

•

•

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

So you end up witha polymer of acrylamide, withoccasional methylene-bisacrylamide cross-links

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 negativecharges due to the SDS impart approximately the same charge-to-massratio for all proteins. Thus, separation iseffected by differences in size.

Reducing agents (e.g., β-mercaptoethanol)to reduce disulfide bonds

An overview of SDS-PAGE electrophoresis

Stacking gel, pH 6.8

Resolving gel pH 8.8

anodecathode

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, theybecome protonated (and therefore zwitterionic) and mostly neutral and relatively immobile. What fractionis in the glycinate form at this pH?

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 encounterthe interface of the stacking gel and the resolving gel.

The key is that the proteins cannot move past theCl− ions. To do so would put them in a region of highconductance 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.

The resolving gel has a higher pH (8.8), which leadsto a higher proportion of glycine molecules becomingdeprotonated and thus negatively charged. Theseglycinate ions overtake the proteins due to theirsmall size. The sieving characteristics of the gelcan now separate proteins on the basis of size.

But the need to pour stacking gels may be obviated: