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Determination of intracellular CMP-sialic acid levels in CHO cells

CK Chan-Yeong Kwak
SP Seung-Yeol Park
CL Chung-Geun Lee
NO Nozomu Okino
MI Makoto Ito
JK Jung Hoe Kim
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The concentration of intracellular CMP-Neu5Ac in CHO cells was examined as previously described36. Briefly, 1.0 × 106 cells were lysed in cold 75% (v/v) ethanol using a cell disruptor. The soluble fraction was separated using centrifugation at 13,000 rpm at 4 °C for 10 min. The lyophilized CMP-Neu5Ac was dissolved with 120 μl of 40 mM phosphate buffer (pH 9.2), followed by centrifugation. The supernatant was filtered using a centricon (MWCO, 10,000) and subjected to CarboPac PA-1 column (Dionex, Sunnyvale, CA). CMP-sialic acid was determined by Abs260 absorbance detector (model 486; Waters). The concentration of intracellular CMP-Neu5Ac was normalized to cell number.

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