Isoelectric focusing

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Isoelectric focusing is an analytical purification technique that separates proteins, electrophoretically, according to their acidic or basic intrinsic charges i.e. their isoelectric point [1].

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 [2].

Isoelectric focusing can distinguish between proteins that differ by as little as one net charge i.e. a pI value of 0.01 [3].

References

  1. Alberts, B. Johnson, A. Lewis, J. Raff, M. Roberts, K. Walter, P. (2008) Molecular Biology of The Cell, 5th edition, New York: Garland Science. Page 521.
  2. Berg, J. Stryer, L. Tymoczko, J. (2007) Biochemistry, 6th Edition, New York: W.H Freeman and Company. Page 73.
  3. Berg, J. Stryer, L. Tymoczko, J. (2007) Biochemistry, 6th Edition, New York: W.H Freeman and Company. Page 73.
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