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Lab Times

1-2015

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47

W

hen classifying the most arche-

typal life science techniques,

gel electrophoresis would most

probably rank amongst the top five. Almost

every researcher working with DNA and/or

proteins sooner or later will have to “run a

gel” to analyse his samples. Hence, life sci-

ence researchers that have never performed

a one-dimensional gel electrophoresis aka

a 1D gel electrophoresis are a pretty rare

species.

The Swedish chemist Arne Tiselius ap-

plied electrophoresis already in the 1930s

to separate serum proteins and earned the

Nobel Prize for his invention in 1948. How-

ever, the real success story of gel electro-

phoresis took off in 1964 when Leonard

Ornstein and Baruch J. Davis, then working

at the Mount Sinai Medical Center in New

York, published two seminal papers describ-

ing a new, improved electrophoresis tech-

nique, which they termed disc(ontinuous)

electrophoresis. Since Ornstein and Davis

used polyacrylamide gels and kept the pro-

teins in the native state, it is also referred to

as the native disc polyacrylamide gel elec-

trophoresis or simply native disc-PAGE.

Back then, life science research was

rather adventurous. The two PAGE-pio-

neers built their electrophoresis apparatus

using carbon rods from old flash light bat-

teries as electrodes and rumours tell, that

they had to “run from the basement of the

hospital to the roof in order to get enough

sunlight ... to aid the catalysis of polymer-

ization of the stacking gels” (Reisfeld and

Williams,

This week’s citation classics

, 1981,

6, 95).

Round-edged electrophoresis tanks

Though a little sport hurts no body dur-

ing a long day in the lab, the times of doubt-

ful homemade electrophoresis chambers

and rusty bulldog clips to fix the gel cas-

settes are (hopefully) in the past.

Modern 1D gel electrophoresis systems

have turned from ugly, square-angled, grey-

coloured plastic boxes with wobbling elec-

trodes into benchtop beauties that may

even please the legendary designer and

enthusiast of rounded edges, Luigi Colani.

The basic concept of native disc-PAGE, how-

ever, has not changed in the last 50 years.

It is still based on the idea that the elec-

trophoretic resolution of charged proteins

is considerably enhanced in a biphasic gel-

buffer system consisting of stacking and re-

solving gel, which differ in pore size, i.e.

acrylamide concentrations, as well as in pH,

ionic strength and anions used in the elec-

trode solutions. The classical system utilises

a Tris-chloride-glycine buffer system com-

posed of Tris-glycine (running buffer) and

Tris-chloride (stacking and resolving gel,

anode buffer); the pH is kept at 6.8 in the

stacking gel in contrast to 8.8 in the resolv-

ing gel and the electrode solutions.

Soon after a voltage is applied, the chlo-

ride ions, already present in the stacking

gel, will take the lead in the race towards

the positively charged anode, while the only

slightly charged glycine ions (pK

A

: 6), flow-

ing in from the cathode buffer, will lag be-

hind.

Protein stacks

The voltage gradient established be-

tween chloride (leading ion) and glycine

(trailing ion), squeezes the proteins run-

ning between leading and trailing ions, to

form a thin protein staple that continuous-

ly moves isotachophoretically through the

wide pores of the stacking gel to the inter-

face of stacking and resolving gel.

When entering the close-meshed resolv-

ing gel, the tightly packed proteins are soon

passed by the smaller and thus faster mov-

ing glycine ions, which run together with

chloride in front of the proteins towards the

anode. On their way through the meander-

ing pores of the resolving gel, the individu-

al proteins of the staple are separated zone-

electrophoretically, according to their size

and electrophoretic mobility.

The invention of the native disc-PAGE

was a milestone in protein research, how-

ever, the method has some shortcomings.

Native proteins can form, for example, er-

ratic moving complexes or may be positive-

ly charged and migrate in the wrong direc-

tion. In 1970, the young Swiss scientist Ul-

rich Karl Lämmli, then working on the bac-

teriophage T4 at the Laboratory of Molec-

ular Biology in Cambridge, had the simple

but brilliant idea of adding sodium dodecyl

sulfate (SDS) to the buffer system.

SDS denatures the proteins and binds

to them in a constant ratio to form evenly

charged, ellipsoidic SDS-protein complex-

es, which are separated in the resolving gel

solely according to their molecular weight.

SDS-PAGE has since become the standard

protein electrophoresis method and is al-

most synonymous with 1D gel electrophore-

sis. SDS-PAGE has stood the test of time and

recent modifications, especially in pre-cast

gels, are limited to variations of the trail-

ing and leading ions (for example, MES or

MOPS instead of glycine and acetate instead

of chloride) and a neutral operating pH.

Only slight variations

While SDS-PAGE is almost exclusively

performed in vertical gel chambers, molec-

ular biologists prefer horizontal submarine

electrophoresis units equipped with aga-

rose or poly acrylamide gels for the elec-

trophoresis of nucleic acids. Submarine gel

boxes are basically rectangular plastic con-

tainers with a cavity on each side connected

by a bridge. The electrodes are mounted at

the bottom of the cavities and coupled to a

jack in the safety lid. The gel is placed onto

the bridge and drowned in electrophoresis

buffer, usually Tris-acetate-EDTA (TAE) or

Tris-borate-EDTA (TBE).

Connecting the electrodes to a power

supply and applying a DC current to the

gel will force the negatively charged DNA

to migrate towards the anode in a size-de-

pendent manner. However, this only holds

true for DNA fragments smaller than ap-

prox. 50 kbp, since long DNA cannot be sep-

arated in a gel applying a constant electric

field. Separating long stretches of DNA re-

quires a pulse field electrophoresis (PFGE)

system that creates an unsteady, periodical-

ly-changing electric field.

PFGE systems are pretty sophisticated

devices, with one type using, for example,

free rotating electrodes to establish a pulsed

electric field. But after all, they still exploit

the same basic electrophoretic principles

that were pioneered by the inventors of gel

electrophoresis some 50 years ago.

Harald Zähringer

Product survey: 1D Gel Electrophoresis systems

Electrophoretic

Race-Tracks

Though discontinuous gel electrophoresis of proteins turned fifty last year,

it is still one of the most vital protein separation methods.

Gel-shit happens

even with stylish, round-

eged gel electrophoresis systems.

Photo: Srimoye Banerjee/Temple University