AGAROSE-SEPHADEX - A NEW IMPROVED MATRIX FOR PREPARATIVE FLAT-BED ISOELECTRIC-FOCUSING

A new, simple procedure is described for the preparation of a gel matrix for preparative isoelectric focusing (IEF) [of proteins], 0.5-1% Agarose of IEF grade is incorporated with 2.5% Sephadex G-200 Superfine (SF) and 3% Pharmalyte, yielding a uniform and rigid matrix. No gel washing, swelling or degassing is necessary. Optimum gel consistency is quickly and easily obtained since no dehydration of the gel slurry is required. Gel preparation time is reduced to 0.5-1 h and gel handling is simplified. Protein can be easily recovered from the matrix. The pH gradient profiles and resolving properties of agarose-Sephadex were compared with those of Sephadex G-200 SF and IEF-grade agarose. The profiles of agarose-Sephadex and Sephadex matrixes are smoother and extend further in both directions than those for agarose. Focusing in agarose-Sephadex and Sephadex also results in fewer edge effects. The ability to distinguish closely resolved bands in agarose-Sephadex is superior due to its ability to be directly stained and its avoidance of electroendosmotic effects associated with focusing in agarose. Changes in voltage enabled us to follow the development, stability, and decay of the pH gradient. Agarose-Sephadex and Sephadex pH gradients developed more smoothly and remained more stable than those for agarose. This new support system combined the good focusing properties of Sephadex G-200 SF with the simple handling, rapid and uniform gel formation of agarose, while overcoming many of the drawbacks of using either material alone.