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April 2002
Vol. 5, No. 4, p 16.
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Fibrous focusing

opening artSearch algorithms used for peptide mapping experiments rely as much on the isoelectric point (pI, the pH at which the net positive and negative charges balance) and mass range information that comes from the original 2-D polyacrylamide gel as on the peptide fragmentation pattern from the mass spectrometer. After proteins are separated in the first dimension by isoelectric focusing (IEF) gel electrophoresis, the gel is extruded from its glass capillary. Compression in the axial direction during extrusion and possible elongation during manual handling might distort or break the gel, disrupting the accuracy of the pI information when the gel is run in the second dimension.

To address this problem, Jon Klein and his colleagues at the University of Louisville School of Medicine (KY) developed a microporous nonwetting hollow fiber to house the IEF gel matrix, replacing the glass tube (J. Proteome Res. 2002, 1, 41–45). The entire unit (gel and fiber) can be soaked and the proteins run through the fiber during electrophoresis in the second dimension. The researchers used a small-diameter pH electrode to first ensure that the fiber maintained the pH gradient, and then they ran IEF standards through the gel.

Getting satisfactory pH gradient and resolution results, the team then ran a series of control proteins through the gel to see how well they resolved and whether they would successfully pass from the IEF matrix into the denaturing gel. For comparison, they performed the same experiment with a standard IEF gel in a glass tube. Both 2-D gels had similar protein spot patterns, but the margins of the spots from the hollow fiber gel were better defined. The researchers also found that maintaining the nonwetted feature of the gels allowed the gels to have an extended shelf life (up to 10 months). The improved shelf life should greatly reduce the cost of running 2-D gels.

RANDALL C. WILLIS

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