Other options For The Preparation Of Gel Electrophoresis

As the ethidium bromide causes cancer and its risky to use ethidium bromide for staining purpose. There are many other options for staining that are safe. We can use methyl blue, SYBR green, Crystal violet and SYBR gold.

If we talk about methyl blue and Crystal violet than the advantage of these agents is that we don’t have to expose the molecules in front of UV in order to visualize them. The UV light is responsible for causing skin cancer.

 By using the agents the chances of mutation in the bands of DNA is reduced. The sensitivity of methyl blue and crystal violet is less as compared to ethidium bromide. While, SYBR green and SYBR gold are more sensitive than ethidium bromide.

The molecules coated with SYBR green and SYBR gold are needed to exposed to UV light and they are also not economic. There are also many other dyes that can be used for the same process but they do not provide desired results.

These dyes are also not added during the process of gel electrophoresis and they are added when the process is completed. Although ethidium bromide has disadvantage but still it is in use as it is easy to use and it is also economic. It is necessary to handle with care and its proper disposal is also necessary.

Loading dye:

There are three purposes of the use of loading dyes. One purpose is that we want to increase the weight of the sample and we add loading dye in order to increase the weightage of the sample.

We increase the weight of the sample in order to properly settle down the sample into the wells created by the comb. It also serves the function of the staining dyes as it produces color on loading and we become sure that our sample has been loaded successfully.

Another purpose of this dye is that as it moves in the gel and we can find out that how much distance has been travelled by the molecules of DNA. These are the three purposes for which we are using loading buffer.

 

 

Nucleic acids:

The DNA contains the negative charge because of the presence of phosphate group and this charge causes the movement of DNA molecules towards the negative electrode. The movement of DNA molecules depend on their size and their circular shape. Multiple bands are obtained if the DNA fragments are circular in shape.

The migration of DNA molecules depends their shape. If the strands of the DNA and RNA molecules are single than they can convert themselves into complicated shapes by turning over. As these molecules are present in complicated shape so the reagents that are used to break hydrogen bonds, they can convert them again into their complicated shapes.

In case of DNA and RNA their gel electrophoresis is carried out by using Agarose gel. The electrophoresis of RNA is performed to check either the DNA has contaminated the RNA or not and either RNA degraded or not. If the RNA is degraded than smearing of bands.

Proteins:

The proteins are different than the nucleic acid in the case that they have complex shapes and different charges. In case of polyacrylamide gel they do not move at the uniform speed. If the different detergents like SDS are used than in the presence of these detergents the proteins are denatured.³

The different detergents are used to imply negative charge on proteins and so they have same charge. The denatured proteins are like rods but those proteins that are not denatured have complex shape. In order to analyze the proteins we use SDS-PAGE.

Nanoparticles:

The technique of gel electrophoresis is also used to separate nanoparticles. The metallic and metal oxide nanoparticles can be separated by using this method. The separation of nanoparticles in on the basis of surface chemistry, size and shape.In the separation of nanoparticles in the gel is on the basis of size.

Results:

As in case of the analysis of proteins there arises the issue of low movement and small pore size. In order to overcome this issue we use Agarose gel for this process. The polyacrylamide gel is not suitable for this purpose. In order to analyze large proteins we use SeaKem gold gel. The Titin protein from the rabbit was analyzed by using this process.

When the proteins are separated, every dot on the gel represents the unique protein. As every dot is representing a protein and there are many dots present in the gel and it is impossible to analyze each spot. The screening of proteins is impossible in this way so we can use different software in order to analyze the proteins.

Factors affecting:

There are many factors that affect the process of gel electrophoresis. These factors are as follows:

·   The charge that is present on the particles affects the process of gel electrophoresis.

·   The molecular weight of the particles also affect this process as the molecules having the large size are easily separated and the molecules having small size are difficult to separate.

·   The different shapes of the molecules also affect this process as the proteins have primary, secondary and tertiary structures and these different levels of the structures make the process complicated.

·   The ph is also responsible for affecting the process and the ph of the solution is maintained by the use of buffer.

·   The electric field that is applied also affects the process and the electric field is applied to allow the movement of molecules towards the electrodes having the opposite charges as the molecules having the positive charge will move towards the electrode having negative charge and the molecules having the negative charge will move towards the electrode having the positive charge.

·   The viscosity of the solution also affects the process. If the solution is more viscous than it will be easy to separate the molecules and if the solution is less than the separation of the molecules becomes little bit tough.

·   The temperature also affects the solution as the high temperature causes the breakdown of proteins.

Effect of temperature:

The temperature and heating has effect on the sample preparation. The Titin protein of the rabbit was destroyed in the presence of high temperature [13]. If the temperature is kept at 100C than we observe more breakdown. If the temperature is reduced to 60C than less breakdown is observed.