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In biochemistry, denaturation is a process in which proteins or nucleic acids lose folded structure present in their native state due to various factors, including application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), agitation and radiation, or heat. [3]
In the earliest forms of denaturation mapping, DNA was denatured by heating in presence of formaldehyde [1] or glyoxal [3] and visualized using electron microscopy. Dyes that selectively bind to double stranded DNA like ethidium bromide could be used to monitor the extent of denaturation.
The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. [1] The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity.
In the less extensive technique of equilibrium unfolding, the fractions of folded and unfolded molecules (denoted as and , respectively) are measured as the solution conditions are gradually changed from those favoring the native state to those favoring the unfolded state, e.g., by adding a denaturant such as guanidinium hydrochloride or urea.
Although stress response pathways are mediated in different ways depending on the stressor involved, cell type, etc., a general characteristic of many pathways – especially ones where heat is the principal stressor – is that they are initiated by the presence and detection of denatured proteins. Because conditions such as high temperatures ...
Nucleic acids are often denatured by including urea in the buffer, while proteins are denatured using sodium dodecyl sulfate, usually as part of the SDS-PAGE process. For full denaturation of proteins, it is also necessary to reduce the covalent disulfide bonds that stabilize their tertiary and quaternary structure , a method called reducing PAGE.
Denaturation midpoint of a protein is defined as the temperature (T m) or concentration of denaturant (C m) at which both the folded and unfolded states are equally populated at equilibrium (assuming two-state protein folding).
Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane. Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility.