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Protocol for pH-gradient chromatofocusing of the native and desialylated human apo-transferrin

This protocol describes the method used for the separation of the native human apo-transferrin from the desialylated apo-transferrin using low-pressure pH gradient ion exchange chromatography. The separation is performed using specialized pH gradient ion exchange chromatography buffers, pISep (CryoBioPhysica, Inc.). The mixture of desialylated apo- transferrin and native apo-transferrin is dissolved in the start buffer (pISep A, pH = 8) and injected onto two HiTrap Q HP (GE Healthcare) anion exchange chromatography column connected in a series. Elution is done by a single step linear gradient (0 – 100 % pIsep B, pH = 4) using ÄKTA Start FPLC system (GE Healthcare). Protein concentration in the eluate is monitored by measuring absorbance at 280 nm and the protein fraction recovery can be calculated by integration over the chromatogram surface area. After separation, the pH value of each fraction containing eluted protein is measured, corresponding to the approximate protein pI value. The observed pI values for the native and desialylated apo-transferrin sialoforms differ significantly and hence can be fully separated.

transferrin, sialoforms, FPLC, chromatofocusing, pISep buffers

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