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Purification of Insulin on UNO S1 Column, Rev A


Insulin is a peptide hormone which stimulates anabolic reactions for carbohydrates, proteins, and fats resulting in lowered blood glucose. It is used therapeutically for treatment of diabetes mellitus and was the first recombinant protein on the market. Although insulin is well characterized and understood, it is often difficult to purify because of the harsh pH and denaturing conditions needed to solubilize and disaggregate the protein. A comparison of the UNO S1 (Bio-Rad catalog # 720-0021) and a popular monodispersed cation exchange column showed that it was advantageous to use the UNO S1 column for small scale insulin separation or analysis.


Experimental
The insulin used in this study was an acid precipitate in suspension. The insulin was solubilized in acidic buffer with urea. The insulin sample was applied separately to an UNO S1 and a monodispersed cation exchange column and eluted by linear gradients. Fractions were collected and neutralized with ammonium hydroxide to pH 7.0. Due to the viscosity of the buffers, both columns were run at half the maximum recommended flow rates. The separation performed on the UNO S1 was run at 3 ml/min (Figure 1). The separation performed on the monodispersed cation exchanger was performed at 1 ml/min (Figure 2). Fractions 4350 and fractions 3034 were pooled from the separation performed on the UNO S1 and the monodispersed column respectively and analyzed by reversed phase HPLC. Analysis performed on Hi-Pore RP-304 reversed phase column (Bio-Rad catalog # 125-0551). The products from both columns were 98% pure. Figure 3 shows the HPLC chromatogram of the pooled fractions from the UNO S1 column after subtraction of the background UV from the buffer.


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