Gel Electrophoresis and alternative tracking dye.
GEL ELECTROPHORESIS is the general and most used method, for the separations, identification and analysis of nucleic acids and proteins. This is also use for determining different fragments of DNA and their weight.
Biomolecules always have a positive or negative ion associated with them. When these molecules are placed in an electric field, they move towards the opposite electrode of their net charge. This happens due to electrostatic interactions. The movements of molecules in the electric field depends upon the net charge, charge to mass ratio, molecular weight and shape of the molecule.
The principle upon which the gel electrophoresis works is related to the equation i.e. v=E.q/f where v is the velocity of charged particle, f is the fractional co-efficient, q is the charge o sample and E is the strength of the electric field. So it is stated that the mobility of charged particle in electric field is equal to the velocity per unit of electric field and measured in q/f ratio. The phenomena is based upon molecular sieving and electrophoretic mobility.
Gel electrophoresis have two types. PAGE and Agarose gel electrophoresis. As compared to agarose electrophoresis, PAGE is of high resolving power, relatively invert, physically stable and have the ability to accept relative large sample size.
The gel of PAGE is made up of Acrylamide, N,N'-methylene bus-acrylamide, buffer and initiation catalyst( TEMED and Ammonium per sulphate). The gel is made according to slab or column setups. The apparatus for both setups are usually same and the only difference is of size of glass plates used,
Sample is loaded in the wells of the gel, when the gel is prepared. Tracking dye is used to trace the sample in the gel. Mobility of ample is due to the electric current supplied to the setup. The alcoholic sugar which is trihydroxyl in nature is called as glycerol. In lipid and carbohydrate metabolisms, glycerol is an intermediate. It acts as a solvent, pharmaceutical agent and sweetening agent.
Gel electrophoresis is a technique used to separate the DNA fragments in accordance to their size. Tracking dye is used to observe the movement of different fragments of DNA in gel. Tracking dye contains a high density reagent. This reagent can be glycerol. Glycerol increases the density of sample such as DNA, and allow the sample to settle at the bottom of the wells of the gel.
The substances used instead of glycerol are, Sucrose, Ficoll 400. These also reacts with the sample and increase its density. Bromophenol blue is used in tracking dye in gel electrophoresis. It is also use as an industrial dye, indicator and staining agent.
Bromophenol blue is a sodium salt. In gel electrophoresis, it is used in the tracking dye because, at moderate pH it is slightly negatively charged thus it moves in the same direction as that of DNA., so it traces the movement of DNA in gel. Also it changes from yellow to blue colour when pH of the sample from 3.6 changes to 4.0, hence it act as tracer marker in gel electrophoresis.
Bromophenol blue (BPB) is regularly used as a dye marker to find out an ion front in the electrophoretic techniques. However, all the chemical, physical and toxicological properties of BPB have not yet been thoroughly investigated. Material safety data sheet of all standard companies providing BPB are advised that due care be exercised when handling this material as it may cause irritation with redness and pain. It is a well known fact that colorants from synthetic sources can be harmful and cause allergies.
Thus, the present study is aimed in a preliminary manner, to find an additional/alternative tracking dye from natural sources to replace the synthetic BPB dye. Bixa orellana L., commonly known as annatto, yielding orange to red colour dye from its pericarp, was tested for its potential as a tracking dye. This dye has characteristics similar to those of BPB and shows no interference with any of the test proteins. The utility of this dye was tested using proteins that exhibit different physicochemical properties and compared with other commonly used staining methods as well as Western blot methods. The studies show that the pigment from B. orellana L. can be used as a tracking dye in place of BPB.
Bromophenol blue is a tetrabromophenol sulfonaphthalein, widely used as a 'tracking dye' by the scientific community. It is an acid-base indicator whose useful range lies between pH 3.0 and 4.6. Since BPB carries a slight negative charge at moderate pH, it will migrate in the same direction as DNA and protein in a gel and thus can be used as a marker ion front . However, to the best of our knowledge all the chemical, physical and toxicological properties of BPB have not yet been thoroughly investigated and Material Safety Data Sheet (MSDS) of all standard companies providing BPB are advised that due care must be exercised when handling this material. BPB may cause irritation with redness and pain when it comes in contact with the skin.
In case of accidental inhalation, it may cause irritation to the respiratory tract. Symptoms may include coughing and shortness of breath. Furthermore, it may cause pain and irritation in the cornea when it comes in contact with the eye.
The recent researches advocate the use of natural dyes instead of BPB as tracking ion front in electrophoretic techniques. To conclude, we have shown that annatto dye, a yellow-coloured pigment from the tropical plant B. orellana L., can be used as a tracking dye for routine SDSPAGE of proteins instead of BPB.
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|Date:||Nov 22, 2015|
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