APPLICATION OF CHEMICALS

APPLICATION OF CHEMICALS NaturomicsWorld-compressed

Amido black: This is used to stain proteins. This can be used as an alternative for Coomassie blue.

Ammonium persulfate (APS): This is used as an initiator in the polymerization of polyacrylamide.

Beta-mercaptoethanol: This is used to reduce the disulfide bonds, thereby denaturing the secondary structure of the proteins.

Bromophenol blue: This is used as a tracking dye during protein and nucleic acid gel electrophoresis.

Cesium chloride: This is used in the purification of plasmids by equilibrium centrifugation.

Chloroform:

This is used as a deprotease agent in combination with isoamyl alcohol in the ratio 24:1. Isoamyl alcohol is an antifoaming agent. Chloroform is also used with phenol during nucleic acid extraction. Chloroform extraction removes proteins as well as phenolic residues present in the nucleic acid preparation.

Coomassie blue: This is used to stain proteins. This can be used to detect only microgram levels of proteins.

Diethylpyrocarbonate (DEPC): This is an RNAse inhibitor used to inactivate RNAses during RNA extraction.

Ethanol: This is used for precipitating nucleic acids. RNA needs 2.5 volumes, and DNA needs 2 volumes. In the presence of 0.17 M NaCl, ethanol will be added.

Ethidium bromide:

This is used to stain the nucleic acids. The substance contains a polymer group that intercalates between the stacked bases of DNA. At 260 nm, UV irradiation is absorbed by DNA transmitted at 590 nm in the red-orange region of the visible spectrum. Ethidium bromide can be used to detect both single- and double-stranded nucleic acids (both DNA and RNA). However, the affinity of the dye for Single-stranded nucleic acid is relatively cheap, but the fluorescent yield is poor. It is possible to use as little as one ng detected by ethidium bromide staining.

Formamide: This is used as a denaturing agent in RNA gel electrophoresis and in hybridization.

Glycerol: This is used in gel loading buffers to increase the density of the samples. This facilitates the samples.to settle at the bottom of the wall.

Heparin: It is an RNase inhibitor.

Hydroxyquinoline: This is an antioxidant used with phenol to prevent the oxidation of phenol. This is also act as RNAse inhibitor and a weak chelating agent.

Isopropanol: This is used for precipitating DNA and plasmids. This has the advantage that the volume of the liquid to be centrifuged is smaller because only an equal volume is added in the presence of 0.3 M sodium acetate.

Tetramethyl ethylene diamine (TEMED): This is used as a catalyst during the polymerization of polyacrylamide.

Phenol or chloroform:

The standard way to remove proteins from nucleic acid is to extract once with phenol, once with a 1:1 mixture of phenol and chloroform, and once with chloroform. The process of deproteinization is efficient when two organic solvents are used. Though phenol denatures proteins efficiently, it does not. It completely inhibits RNase activity and acts as a solvent for poly(A)-containing RNA stretches. Both these problems can be overcome by using the above mixture.

Phenol: This is used as a deproteinizing agent during nucleic acid extraction. 8-Hydroxyquinoline, to a final product, an antioxidant, a partial Rnase inhibitor, and a weak metal ion chelator are added to the phenol concentration of 0.1%.

Sodium dodecyl sulphate (SDS):

This is used as a denaturing agent in RNA gel electrophoresis and in polyacrylamide gel electrophoresis It is also used to rupture the cell membrane. Being an anionic detergent, SDS attaches an anionic group (negatively charged) at regular intervals along the polypeptide chains, thereby making the separation only on the basis of mass in SDS-PAGE.

Tichloroacetic acid (TCA): This is used to precipitate proteins and nucleic acids. During protein precipitation, the proteins are denatured, thereby turning the solution into a suspension.

Written by :-
Vishwajit
MSc.Biotechnology

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