Isoelectric focusing: Difference between revisions
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Isoelectric focusing is an analytical purification technique that separates [[Protein|proteins]], electrophoretically, according to their acidic or basic intrinsic charges i.e. their [[ | Isoelectric focusing is an analytical purification technique that separates [[Protein|proteins]], electrophoretically, according to their acidic or basic intrinsic charges i.e. their [[Isoelectric point|isoelectric point]]. | ||
A [[Polyacrylamide|polyacrylamide]] gel apparatus containing a [[ | A [[Polyacrylamide|polyacrylamide]] gel apparatus containing a [[PH|pH gradient]] is used. The [[PH|pH]] gradient is established by the subjection of a [[Buffer|buffer]]-like mixture of many [[Polyampholytes|polyampholytes]], each with a different [[Isoelectric point|isoelectric point]], to [[Electrophoresis|electrophoresis]]. The [[Protein|protein]] sample is then loaded and a suitable [[Voltage|voltage]] is applied to the electric field. The [[Protein|protein]] migrates to its characteristic [[Isoelectric point|isoelectric pH forming]] a band and, once reached, its electrophoretic mobility becomes zero; movement in the positive or negative direction is inhibited. | ||
Isoelectric focusing can distinguish between [[Protein|proteins]] that differ by as little as one net charge i.e. a [[ | Isoelectric focusing can distinguish between [[Protein|proteins]] that differ by as little as one net charge i.e. a [[Isoelectric point|pI]] value of 0.01 <ref>Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. Walter, P. (2008) Molecular Biology of The Cell, 5th edition, New York: Garland Science.</ref>. | ||
=== References === | |||
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Revision as of 22:06, 9 January 2011
Isoelectric focusing is an analytical purification technique that separates proteins, electrophoretically, according to their acidic or basic intrinsic charges i.e. their isoelectric point.
A polyacrylamide gel apparatus containing a pH gradient is used. The pH gradient is established by the subjection of a buffer-like mixture of many polyampholytes, each with a different isoelectric point, to electrophoresis. The protein sample is then loaded and a suitable voltage is applied to the electric field. The protein migrates to its characteristic isoelectric pH forming a band and, once reached, its electrophoretic mobility becomes zero; movement in the positive or negative direction is inhibited.
Isoelectric focusing can distinguish between proteins that differ by as little as one net charge i.e. a pI value of 0.01 [1].
References
- ↑ Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. Walter, P. (2008) Molecular Biology of The Cell, 5th edition, New York: Garland Science.