Importance

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Abstract

Significantly higher yield and better resolution in pI gels are obtained by creating traps having two or more layers of gel containing closely stepped immobilized pH buffers. Proteins move from a pH at which they are negatively charged towards an anode at which they are positively charged. Discrete regions containing immobilized pH buffers trap the proteins when the immobilized buffer pH and the protein pI are approximately the same. The protein is trapped within the second layer and not on the surface of or interface of the second layer. Significantly higher yields with better resolution can be obtained through the use of layered sample application gels prior to isoelectric focusing. Layered plugs are prepared with a range of immobilized pH buffers ranging, for example, over 2 pH units, with steps of 0.05 or 0.1 pH units. An array of multilayered plugs wherein each plug has different pH increments is also provided. The array can be used to isolate and trap a variety of proteins having different isoelectric pHs during a single run. Another embodiment provides plugs having at least three layers; a gate layer, a trap layer, and an exit layer. Another embodiment includes adding a carrier ampholytes to running buffers and or adding thiol containing reducing agents to reduce current and improve resolution and collection efficiency.

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First Claim