Gel Electrophoresis

Introduction:

The molecules consist of both positive and negative charges and when we place these charges in an electric filed than these charges move towards the opposite poles like positive charges move towards negative poles and negative charges move towards positive poles. The movement of charges towards opposite poles is due to electrostatic attraction.

Electrophoresis is defined as the movement of charges towards opposite electrodes when they are placed in electric field. The movement of different molecules is different and this movement depends on many factors like temperature, viscosity, porosity, charge/mass ratio and net charge [Sheehan d; 2000].

In gel electrophoresis the small molecules move fast as their size is small and large molecules move slowly as they have large size. In order to separate proteins we can separate them on the basis of charge. The nanoparticles can also be separated by using this technique.

For analytical purposes we can also use this technique. The DNA is amplified by using PCR technique and that amplified DNA can be analyzed with the help of this technique. The DNA that is used in Sothern blotting also involves the use of gel electrophoresis.

The major challenge in proteomics is the quantitative analysis of proteins. There are different methods which can be used for the analysis of proteins like proteolysis of protein samples can be carried out. The large fragments of DNA can be analysed but the small fragments are not analysed easily and the method of gel electrophoresis can be used for the detection of such type of small fragments.

The Agarose gel is used in horizontal dimension and polyacrylamide gel is used in vertical dimension. The microtiter well plates are used in this process as the wells are filled with sample. This technique can be carried out at large scale by using tanks. A tank can accommodate 1000 to 2000 samples.

We use vertical type of gel electrophoresis for the separation of proteins. The size of DNA depends on the number of base pairs that are present. The negative charge that is present on DNA is due to the presence of phosphate group.

When we observe the secondary structure of DNA than the position of phosphate groups is outside the molecule that impart negative charge to the DNA. In case of proteins denaturation is required but in case of DNA the step of denaturation is not required.

The movement of DNA in the gel depends on the length of DNA. In order to perform the electrophoresis of DNA the Agarose gel is required if the size of DNA molecules is larger than 500bp and the length of DNA must be determined.

If the length of DNA is less than the 500bp than the Agarose gel is not useful as it has large pore size so for such case the polyacrylamide gel is used which has small pore size. When the molecules of DNA are separated we stain them by using different agents like ethidium bromide and then exposed to UV light in order to see the molecules of DNA.

In order to study RNA we can also use the process of gel electrophoresis. As the RNA is different from DNA so when we are performing the gel electrophoresis of RNA the process is different from the gel electrophoresis of DNA.

As the RNA contains the secondary structure so this structure must be effectively destroyed and base pairing should be removed by the pretreatment process but in case of DNA the pretreatment process is not required. The base pairing will affect the movement of RNA in the gel.

If we want to measure the movement of DNA and RNA in the gel than we must know the size of the fragments of DNA and RNA. When we denature the RNA and DNA they are converted into the single strands and the denaturation of DNA can be easily done with the alkali.

When the denatured strands of DNA or RNA than under certain conditions that will support the annealing of the strands than strands will join together by forming hydrogen bonds. This feature of nucleic acids will help them in different techniques like molecular biology.

This feature is also used in laboratory techniques and we use this in order to find the sequences of DNA and RNA. When the single strands are present in gel than the radiolabelled strands are allowed to move in the gel than the conditions for the annealing of the strands are provided and the double stranded molecules are formed.

Principle:

Electrophoresis is a mixture of charged molecules that are placed in an electric field and the strength of electric field is E, in the presence of which charges move towards opposite poles. The different molecules move with different speed as they have different physical properties.

The velocity of molecules is v and frictional coefficient is f and net charge is q. The term frictional coefficient is defined as the resistance that is faced by the molecules during movement and this depends on factors like; porosity, viscosity of buffer and the mass of molecule.

In order to determine the net charge we have to determine the total positive and negative charges that are present in a molecule. In case of proteins the charges are due to presence of amino acid groups and the groups that arise from phosphorylation. On DNA the negative charge is due to the presence of phosphate group and the charge is uniformly distributed.

The speed of molecules increases when the charge increases and also when the strength of electric field increases. If the molecules have same charge than the separate out on the basis of difference in frictional coefficient and those molecules which have same shape they differ on the basis of net charge.

Base for separation:

In order to separate out proteins we can use two properties of proteins which are as follows:

·         First property is that we can separate the proteins on the basis of their charge as proteins has positive charge on them due to the presence of amino groups.

·         Second property is that we can separate the proteins on the basis of their size or mass as the proteins of higher mass can be separated easily and proteins of the low mass can be separated with difficulty.

·         When we are using the method of 2D gel electrophoresis than there are chances that charge or mass of proteins can observe any change.

Isoelectric point:

As the proteins contain the negative charge on them and there is a value of ph at which there is no net charge on the protein. Above the isoelectric charge the protein contains the negative charge due to the presence of amino groups and in the gel the proteins move towards the anode as the anode contains the positive charge.

When the protein is below the isoelectric point than it contains the positive charge and it tends to move towards the cathode as it contains the negative charge.

Isoelectric focusing:

At the isoelectric point there is no charge on the proteins and if we separate proteins by using the term isoelectric point than this process is known as isoelectric focusing. The requirements for this process are ph and electric potential that are applied to the gel.

When isoelectric point is not reached than the proteins has net charge. Above isoelectric point the proteins has negative charge and below isoelectric point the proteins has positive charge.

If the charge on the proteins is positive than they will move towards the cathode having negative charge. If the charge on proteins is negative than they will move towards the anode having positive charge.

In case of first dimension electrophoresis the movement of proteins will be such that they move along gel and when the isoelectric point of protein reaches they will be accumulated.

Materials:

The materials that are used in this process include:

·         Perspex blocks.

·         CNC machining center.

·         Microtiter well plates.

·         Cutting blades.

·         Different types of gel that can be either Agarose or polyacrylamide or many more.

Gel electrophoresis:

There are many gel networks and types of gel electrophoresis that allow successful completion of many experiments. Many other experiments like blotting and many more are possible because of this technique as it is mechanically stable.

DNA finger printing also involves the use of this technique. The gels that are used in this technique are un reactive and they do not cause toxicity. The gels during this process interfere with the separating molecules and separate them [Adamson; 1997].

Physical bases:

The technique gel electrophoresis involves the separation of molecules on the basis of their size. An electric field is applied in the solution and due to this electric filed the molecules move towards their opposite poles. This electric field creates the negative charge at one pole and positive charge at the other end.

Wells are created in which sample is placed. After that place the gel tray in a chamber that is used for electrophoresis purpose. The power supply is than connected to the chamber and due this the molecules start moving towards the opposite poles and the molecules of small size move with greater speed and molecules of large size move with less speed.

As there are different sized molecules so, we obtain different bands.⁴ The porosity or composition of the matrix of gel depends on the nature of the molecules that are to be separated. When we want to separate those nucleic acids that are large in size than we use Agarose gel. Polyacrylamide is used as a gel but this is a neurotoxin and must be handled with great care.

In case of Agarose uncharged carbohydrates are present. We place the sample in wells and then apply electric field which will move the molecules on the basis of their size as the molecules of small size will move with greater speed and molecules of large size will move with low speed.

If the charges are not uniform than the molecules will be separated on the basis of charge. The molecules that have positive charge they will move towards the electrodes having negative charge and the molecules having negative charge will move towards the electrode having positive charge.

The speed of molecules also depends on their mass as the lighter molecules move faster and heavier molecules move slowly.⁶ if in a single gel many samples are loaded than they create parallel lines. Each line shows the separation of molecules from their original solution.

If the separation of molecules is incomplete than the overlapping bands will be formed and also smearing occurs. The bands from different lines end at the same distance it shows that all of these molecules have same speed. The same speed of the molecules mean that they all have same size.

We can also use different size markers in which molecules of the known size are present. These markers are run on the gel and then they are compared with the unknown sizes in order to determine the sizes. The distance of the band is proportional to the size of molecules.

The disadvantages of the use of gel electrophoresis include: as we pass the electric current in order to allow the movement of molecules this can melt the gel. In this technique we use buffer that maintains the ph as the charges of DNA and RNA are dependent on the use of buffer but the too long use of buffer can exhaust the solution.

In order to find out the molecular weight of any unknown protein we use the technique of SDS-PAGE and this technique also has many limitations. There are many factors that affect the movement of molecules in the gel electrophoresis technique. There are many proteins that migrate in an abnormal way as they don’t have correct charge/mass ratio.