Discontinuous electrophoresis (colloquially disc electrophoresis[a]) is a type of polyacrylamide gel electrophoresis. It was developed by Ornstein and Davis.[2][1] This method produces high resolution and good band definition. It is widely used technique for separating proteins according to size and charge.[3]
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Discontinuous Electrophoresis
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SDS PAGE Principle and the Use of Discontinuous buffer system
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advantage of discontinuous buffer system in SDS and native PAGE is
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Method
In this method, the gel is divided into two discontinuous parts, resolving and stacking gel, both have different concentrations of polyacrylamide.[4] 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 contains glycine. 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 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.[5][6]
See also
References
- ^ a b Ornstein, Leonard (2006-12-16). "Disc Electrophoresis-I Background and Theory". Annals of the New York Academy of Sciences. Wiley. 121 (2): 321–349. doi:10.1111/j.1749-6632.1964.tb14207.x. ISSN 0077-8923. PMID 14240533. S2CID 28591995.
- ^ Williams, Donald E. & Reisfeld, Ralph A. (1964). "DISC Electrophoresis IN POLYACRYLAMIDE GELS". Annals of the New York Academy of Sciences. 121 (2): 372–381. Bibcode:1964NYASA.121..373W. doi:10.1111/j.1749-6632.1964.tb14210.x. PMID 14240536. S2CID 46152998.
- ^ PolyAcrylamide Gel Electrophoresis (PDF). davidson.edu.
- ^ "Principle of Disc Electrophoresis". Archived from the original on 29 June 2016. Retrieved 4 July 2016.
- ^ Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis Author H. R. Maurer Edition 2, illustrated Publisher Walter de Gruyter, 1978 ISBN 3110836203, 9783110836202
- ^ "EXPERIMENTAL TECHNIQUES - Electrophoresis". Retrieved 4 July 2016.
External links
This page was last edited on 4 February 2024, at 20:05