通过醋酸纤维素电泳对人珠蛋白链合成进行定量分析。

Quantitation of human globin chain synthesis by cellulose acetate electrophoresis.

作者信息

Salmon J E, Nudel U, Schiliro G, Natta C L, Bank A

机构信息

Department of Medicine, Cancer Research Center, Columbia University, New York, New York 10032, USA.

出版信息

Anal Biochem. 1978 Nov;91(1):146-57. doi: 10.1016/0003-2697(78)90825-4.

DOI:10.1016/0003-2697(78)90825-4

PMID:9762093

Abstract

We have developed a method for the separation and quantitation of human alpha-, beta-, and gamma-globins utilizing cellulose acetate electrophoresis. The relative rates of synthesis of globin chains in reticulocytes in peripheral blood is determined by: (i) incubating intact cells with [35S]methionine; (ii) preparing globin from the hemolysates; (iii) performing electrophoresis of the globin on cellulose acetate strips; and (iv) autoradiography or direct determination of the radioactivity incorporated into each globin chain. The method is simple and rapid, requires only small amounts of hemolysate (30 micrograms of globin), and provides excellent resolution and reproducible quantitation of alpha-, beta A-, beta S-, and gamma-globin chains for up to 24 peripheral blood samples at one time. Measurements by this method in patients with thalassemia variants and sickle-cell disorders correlate well with analysis of the same samples by carboxymethyl cellulose chromatography. This methodology may permit more widespread analysis of globin synthesis in the thalassemia syndromes and may also be useful in the analysis of globins synthesized from human globin mRNA in cell-free systems.

摘要

我们开发了一种利用醋酸纤维素电泳分离和定量人α、β和γ珠蛋白的方法。外周血网织红细胞中珠蛋白链的相对合成速率通过以下步骤确定：（i）用[35S]甲硫氨酸孵育完整细胞；（ii）从溶血产物中制备珠蛋白；（iii）在醋酸纤维素条上对珠蛋白进行电泳；（iv）放射自显影或直接测定掺入每条珠蛋白链的放射性。该方法简单快速，只需要少量溶血产物（30微克珠蛋白），并且能对α、βA、βS和γ珠蛋白链进行出色的分离，并能同时对多达24份外周血样本进行可重复的定量分析。用该方法对地中海贫血变异型和镰状细胞病患者进行测量，与用羧甲基纤维素色谱法对相同样本进行分析的结果相关性良好。这种方法可能使地中海贫血综合征中珠蛋白合成的分析更广泛地开展，并且在分析无细胞系统中由人珠蛋白mRNA合成的珠蛋白时也可能有用。