A gel-based method for purification of apolipoprotein A-I from small volumes of plasma |
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Authors: | Rachel J. Brace Brie Sorrenson Dmitri Sviridov Sally P. A. McCormick |
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Affiliation: | *Department of Biochemistry, University of Otago, Dunedin, New Zealand;†Baker IDI Heart and Diabetes Institute, Melbourne, Australia |
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Abstract: | We present here a gel-based method for rapid purification of apolipoprotein A-I (apoA-I) from small volumes of human plasma. After isolation of high density lipoprotein from plasma, the apoA-I protein was separated by electrophoresis and the apoA-I band excised from the gel. The apoA-I was then eluted from the gel strip, concentrated, and delipidated ready for use. The structure and function of the gel-purified apoA-I protein was compared against apoA-I purified by the traditional size-exclusion chromatography method. The α-helical content of the gel-purified apoA-I as determined by circular dichroism was similar to chromatography-purified apoA-I. The functional activity of gel-purified apoA-I, as determined by cholesterol efflux assays in primary human fibroblasts and RAW264.7 macrophages, was also comparable with chromatography-purified apoA-I. This method is a valid alternative for apoA-I purification with some advantages over traditional chromatography purification including a much reduced plasma volume requirement, less time and cost, and a higher percentage protein recovery. The method is particularly suitable for applications requiring the purification of apoA-I from multiple human or animal samples of interest. |
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Keywords: | high density lipoproteins gel purification size-exclusion chromatography cholesterol efflux |
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