Howe J G, Hershey J W
J Biol Chem. 1981 Dec 25;256(24):12836-9.
Protein synthesis initiation factor levels are measured in crude cell lysates of Escherichia coli MRE600 by use of a sensitive immunoblotting method. The method involves electrophoretic transfer of protein from sodium dodecyl sulfate-polyacrylamide gels onto nitrocellulose paper and subsequent incubation with a specific antiserum and radioactive iodinated second antibody. The measurement of iodinated antibody attached to known amounts of initiation factor is determined by densitometric scanning of autoradiographs or counting radioactivity in excised protein bands. Linear standard curves over the range 1 to 300 ng of factor are obtained by these methods. Unknown amounts of initiation factor in crude cell lysates are measured accurately; values agree with previous radioimmune assay data. The immunoblotting method serves as an alternative to the radioimmune assay in measuring small quantities of protein in complex mixtures. Immunoblotting enjoys three major advantages: it is simple and rapid to execute; it is sensitive; and it is capable of distinguishing multiple forms of the antigen which separate in the gel system employed.
利用一种灵敏的免疫印迹法，在大肠杆菌MRE600的粗细胞裂解物中测定蛋白质合成起始因子的水平。该方法包括将蛋白质从十二烷基硫酸钠-聚丙烯酰胺凝胶电泳转移到硝酸纤维素纸上，随后与特异性抗血清和放射性碘化二抗一起孵育。通过对放射自显影片进行光密度扫描或对切下的蛋白条带中的放射性进行计数，来测定附着在已知量起始因子上的碘化抗体。通过这些方法，可得到1至300 ng因子范围内的线性标准曲线。粗细胞裂解物中未知量的起始因子可被准确测定；其值与先前的放射免疫测定数据一致。免疫印迹法可作为放射免疫测定的替代方法，用于测量复杂混合物中的少量蛋白质。免疫印迹法具有三个主要优点：操作简单快速；灵敏度高；能够区分在所用凝胶系统中分离的多种抗原形式。
