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Discontinuous electrophoresis (colloquially ''disc electrophoresis'') is a type of
Analysis of C14-labeled proteins by disc electrophoresis
{{electrophoresis Electrophoresis
Discontinuous electrophoresis (colloquially ''disc electrophoresis'') is a type of polyacrylamide
Polyacrylamide (abbreviated as PAM) is a polymer with the formula (-CH2CHCONH2-). It has a linear-chain structure. PAM is highly water-absorbent, forming a soft gel when hydrated. In 2008, an estimated 750,000,000 kg were produced, mainly f ...
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
. It was developed by Ornstein and Davis. This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.
Method
In this method, the gel is divided into two discontinuous parts, resolving and stacking gel, both have different concentrations of polyacrylamide. The one with lower concentration is stacked on top of the one with higher concentration. Discontinuity is based on four parameters: gel structure, pH value of the buffer, ionic strength of the buffer, and the nature of the ions in the gel and electrode buffer. The electrode buffer containsglycine
Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinog ...
. Glycine has very low net charge at pH 6.8 of stacking gel, so it has low mobility. The proteins are separated according to the principle of isotachophoresis and form stacks in the order of mobility (stacking effect). Mobility depends on net charge, not on the size of the molecule. Proteins move towards anode
An anode is an electrode of a polarized electrical device through which conventional current enters the device. This contrasts with a cathode, an electrode of the device through which conventional current leaves the device. A common mnemoni ...
slowly at constant speed till they reach limit of separation gel. Suddenly, frictional resistance increases but glycine is not affected and it passes the proteins and becomes highly charged in resolving zone. Proteins present in homogeneous buffer start to separate based on principles of zone electrophoresis. Now their mobility depends on size as well as charge. pH value rises to 9.5 and net charge increases.
See also
* Affinity electrophoresis *SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 kDa. ...
* Isotachophoresis
References
External links
Analysis of C14-labeled proteins by disc electrophoresis
{{electrophoresis Electrophoresis