Product Survey: 1D Gel Electrophoresis systems

Electrophoretic Race-Tracks
by Harald Zähringer, Labtimes 01/2015



Though discontinuous gel electrophoresis of proteins turned fifty last year, it is still one of the most vital protein separation methods.

When classifying the most archetypal 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 science researchers that have never performed a one-dimensional gel electrophoresis aka a 1D gel electrophoresis are a pretty rare species.


Gel-shit happens−even with stylish, round-eged gel electrophoresis systems. Photo: Srimoye Banerjee/Temple University

The Swedish chemist Arne Tiselius applied electrophoresis already in the 1930s to separate serum proteins and earned the Nobel Prize for his invention in 1948. However, the real success story of gel electrophoresis 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 describing a new, improved electrophoresis technique, which they termed disc(ontinuous) electrophoresis. Since Ornstein and Davis used polyacrylamide gels and kept the proteins in the native state, it is also referred to as the native disc polyacrylamide gel electrophoresis or simply native disc-PAGE.

Back then, life science research was rather adventurous. The two PAGE-pioneers built their electrophoresis apparatus using carbon rods from old flash light batteries 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 polymerization 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 during a long day in the lab, the times of doubtful homemade electrophoresis chambers and rusty bulldog clips to fix the gel cassettes are (hopefully) in the past.

Modern 1D gel electrophoresis systems have turned from ugly, square-angled, grey-coloured plastic boxes with wobbling electrodes into benchtop beauties that may even please the legendary designer and enthusiast of rounded edges, Luigi Colani. The basic concept of native disc-PAGE, however, has not changed in the last 50 years. It is still based on the idea that the electrophoretic resolution of charged proteins is considerably enhanced in a biphasic gel-buffer system consisting of stacking and resolving gel, which differ in pore size, i.e. acrylamide concentrations, as well as in pH, ionic strength and anions used in the electrode solutions. The classical system utilises a Tris-chloride-glycine buffer system composed 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 resolving gel and the electrode solutions.

Soon after a voltage is applied, the chloride 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 (pKA: 6), flowing in from the cathode buffer, will lag behind.

Protein stacks

The voltage gradient established between chloride (leading ion) and glycine (trailing ion), squeezes the proteins running between leading and trailing ions, to form a thin protein staple that continuously moves isotachophoretically through the wide pores of the stacking gel to the interface of stacking and resolving gel.

When entering the close-meshed resolving gel, the tightly packed proteins are soon passed by the smaller and thus faster moving glycine ions, which run together with chloride in front of the proteins towards the anode. On their way through the meandering pores of the resolving gel, the individual 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, however, the method has some shortcomings. Native proteins can form, for example, erratic moving complexes or may be positively charged and migrate in the wrong direction. In 1970, the young Swiss scientist Ulrich Karl Lämmli, then working on the bacteriophage T4 at the Laboratory of Molecular 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 complexes, 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 almost synonymous with 1D gel electrophoresis. SDS-PAGE has stood the test of time and recent modifications, especially in pre-cast gels, are limited to variations of the trailing 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, molecular biologists prefer horizontal submarine electrophoresis units equipped with agarose or poly acrylamide gels for the electrophoresis of nucleic acids. Submarine gel boxes are basically rectangular plastic containers 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-dependent manner. However, this only holds true for DNA fragments smaller than approx. 50 kbp, since long DNA cannot be separated in a gel applying a constant electric field. Separating long stretches of DNA requires a pulse field electrophoresis (PFGE) system that creates an unsteady, periodically-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.




First published in Labtimes 01/2015. We give no guarantee and assume no liability for article and PDF-download.


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