Electrophoresis is the term used for the procedure where under the influence of voltage, a charged particle moves. It is a standard method for separation, identification, DNA restriction fragments analysis and purification. Agarose is basically a linear polymer comprising D-galactose and L- galactose, when it solidifies it forms a matrix like structure which contains pores, these pores allows the sieving of the molecules (eg. DNA). The agarose concentration in the gel decides the separation of DNA depending upon its size. The concentration of agarose when increased results in the decrease of the speed of the molecule movement in the gel and hence  result in better resolution. The smaller DNA molecule moves faster through the gel whereas the bigger fragments are retained on the upper part of the gel as they migrate slowly. The shape of DNA also determines the movements in the gel. The movement of the DNA fragment in the gel also directly proportional to the voltage applied.

PRICIPLE AND THEORY: Electrophoresis is essentially the migration of a charged molecule under the influence of electric field in the direction of cathode or anode depending upon the charge on the molecule. A buffer solution is use to conduct electricity through the whole setup of the agarose gel electrophoresis, the DNA which is negatively charged will migrate through the gel depending upon the size and shape.

In this electrophoresis, the agarose gel is first prepared in the electrophoresis buffer. A certain amount of agarose powder is mixed with the electrophoresis buffer and then heated in the microwave oven so that it completely dissolves and melts. After cooling it for some time it is poured into the casting tray. The casting tray should already have sample comb which will make wells in the gel. After pouring the gel it is allowed to cool and set. After the gel is set the comb is slowly removed and the gel is then set horizontally into the electrophoresis chamber. The electrophoresis buffer is then poured such that it submerges the gel completely. The lid is put on and the current is switched on, the bubbles coming up in the electrophoresis buffer solution tells us that the current is flowing. The DNA is negatively charged so it will migrate towards anode, we can deduce the distance travelled by the DNA in the gel by visually monitoring the distance travelled by the loading dye. Usually bromophenol is used as loading dye (xylene cyanol is also used), these loading dyes migrate at similar rate as that of the DNA molecules.

Procedure:

S₂O₈^2-  + e^-                  SO₄^2- + SO₄^-- ∙

This free radical helps in polymerization. The pore size of the acrylamide gel can be controlled by varying the concentration of the acrylamide in gel. For large pore size low percentage gels are used, these types of gel help separating protein molecules and DNA molecules. in SDS PAGE system mostly 10 or 20 percent gel are used to separate protein. Separation on the basis of size is done as sieving effect is achieved using small pore sized gels. We get better defined bands as the SDS ion mostly destroys the secondary and tertiary structure of the protein and linearize the protein sample and as the SDS-protein complex gets concentrated between the Cl^- ions and glycinate ions.

In Sodium dodecyl sulphate polyacrylamide gel electrophoresis the gel comprises of two types of restrictive gels:

i.                   Stacking gel (pH 6.8)

ii.                 Separating/ resolving gel (pH 8.8)

The pH and the concentration of the two gels are different 

The smaller protein will migrate farther in the gel and the larger protein will be retained on the upper portion. Sodium dodecyl sulphate (Shapiro et al 1967) is basically an anionic detergent and helps in separating molecules on the basis of molecular weight. This technique utilizes restrictive gels and hence we get improved resolution.

These two different layers (separating and resolving) are responsible for separating the polypeptides on the basis of their size. The top layer is the stacking gel which has the wells which are loaded with the samples to be differentiated and the constituent of this gel layer helps in compacting or stacking of the samples this allows us to get better resolution and finer bands. SDS PAGE is discontinuous gel system therefore the gel is poured in two steps. First the resolving gel or separating gel is prepared, gel is formed by polymerization which is initiated by adding ammonium per sulphate to the mix and then addition of TEMED, polymerization occurs quickly after the addition of TEMED therefore everything should be ready for casting the gel on the electrophoresis assembly. After adding the resolving gel water is poured in for forming a smooth level. Within ten to fifteen minutes the gel is polymerized and the water can be removed from the upper layer for casting stacking gel. The stacking gel includes Tris-Cl and SDS with a ph of 6.8, usually the stacking gel is composed of 3% to 5% acrylamide, now after adding Ammonium per sulphate and TEMED the mix is swirled and poured on top of the separating gel. Comb for making wells are placed gently and immediately without bubble formations.

Points to remember while preparing the gel:

Always wear gloves while handling the acrylamide as it is a toxic substance. A gel with particular acrylamide concentration can help in separating certain size range of proteins. In a low percentage gel the larger polypeptides can enter easily and well separated.

Assembly, Loading and Running of Gels:

The assembly should be such that the current flows through the gel, electrophoresis buffer is filled in the tank, this buffer consists of Tris, glycine and sodium dodecyl sulphate. Before adding the buffer the comb should be removed. For loading the samples we may also add glycerol which makes the sample settle into bottom of the wells properly. Anode will attract the proteins as our proteins are negatively charged. The voltage applied is such that it will make the tracking dye migrate towards the end of the gel quickly without overheating the gel. After the tracking dye has migrated to the end of the gel the electrophoresis is stopped and the gel is taken out and kept for staining.

Staining the protein gel:

Coomasssie Brilliant Blue dye with methanol and glacial acetic acid is usually used for staining the polyacrylamide gel. The methanol in the dye precipitates the protein. The dye attaches to the proteins and the washing step following the staining removes the excess dye off. We get protein bands against a clear background after properly staining. Silver staining is used when we want to stain very little amount of proteins.

Mobility: (applied voltage) X (overall net charge on molecule) / (friction on molecule)

The SDS neutralizes the shape of the molecules thereby the separation takes place on the basis of the size or molecular weight.

                                                                    

In general, gel electrophoresis is used to separate the different size of charged molecules(DNA or RNA or protein) by placing them between cathode and anode,these molecules get arranged according to their size and molecular weight.Distance traveled by these molecules from loading site is inversely proportional to the size and molecular weight of the charged molecules.

References:

·        Georgia Institute of Technology,2010 “SDS Page Gel Electrophoresis”(updated on 05/01/2001)Available at <http://ww2.chemistry.gatech.edu/~lw26/bCourse_Information/4581/techniques/gel_elect/page_protein.html> [Accessed on 4th December 2010].

·        Singh B.D., 2004, Biotechnology-Expanding Horizons. New Delhi: Kalyani Publishers.

·        Smith College, Biochemistry Laboratory,2004 “SDS Gel Electrophoresis” Available at< http://www.science.smith.edu/departments/Biochem/Biochem_353/sdspage.html> [Accessed on 4^th December 2010]

Writers: Nazia Tabassum & Abhishek Sharma

   (M.Sc. Student, University of Sussex,UK)