通过多核糖体免疫吸附法纯化大鼠肝脏果糖-1,6-二磷酸酶的编码信使核糖核酸

Purification of mRNA coding for rat-liver fructose-1,6-bisphosphatase by polysome immunoabsorption.

作者信息

el-Dorry H A

出版信息

Biochim Biophys Acta. 1986 Aug 22;867(4):252-5. doi: 10.1016/0167-4781(86)90041-2.

Abstract

The mRNA coding for rat liver fructose-1,6-bisphosphatase, which represents approx. 0.46% of total hepatic mRNA, has been purified to near homogeneity. Polysomes from rat liver were allowed to react with antibodies to rabbit anti-fructose-1,6-bisphosphatase purified by affinity chromatography. The complex was immobilized on a protein A-Sepharose column. After the removal of unabsorbed polysomes, the specific mRNA was eluted and chromatographed on an oligo(dT)-cellulose column. This method gave a 183-fold enrichment of the fructose-1,6-bisphosphatase mRNA to greater than 80% homogeneity as determined by electrophoreses of immunoprecipitated in vitro translation products on polyacrylamide slab gels in the presence of sodium dodecyl sulphate.

摘要

编码大鼠肝脏果糖-1,6-二磷酸酶的信使核糖核酸（mRNA），约占肝脏总mRNA的0.46%，已被纯化至接近均一。使来自大鼠肝脏的多核糖体与针对通过亲和层析纯化的兔抗果糖-1,6-二磷酸酶的抗体发生反应。该复合物被固定在蛋白A-琼脂糖柱上。去除未吸附的多核糖体后，洗脱特异性mRNA并在寡聚（dT）-纤维素柱上进行层析。通过在十二烷基硫酸钠存在下在聚丙烯酰胺平板凝胶上对免疫沉淀的体外翻译产物进行电泳测定，该方法使果糖-1,6-二磷酸酶mRNA富集了183倍，均一性大于80%。