Immunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins.
Immunoautoradiographic detection of proteins after electrophoretic transfer from gels to diazo-paper: analysis of adenovirus encoded proteins.
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从凝胶电泳转移到重氮纸后蛋白质的免疫放射自显影检测：腺病毒编码蛋白质的分析。
DOI：
10.1073/pnas.78.1.177
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发表时间：
1981
影响因子：
11.1
通讯作者：
Brackmann,K
中科院分区：
文献类型：
--
作者：
Symington,J;Green,M;Brackmann,K
我们描述了一种方法，通过标准的一维Na-DodSO4/聚丙烯酰胺凝胶电泳或O'Farrell二维凝胶电泳对复杂的蛋白质混合物进行分离，然后高效快速地电泳转移到重氮苄氧甲基或重氮苯基硫醚纸上，并通过免疫自放射照相进行分析。用感染2型腺病毒的人KB细胞的蛋白提取物说明了该方法。当使用[35S]-蛋氨酸标记的蛋白质时，蛋白质从凝胶中转移而不降低分辨率，并增加了放射自显影检测的灵敏度。当未标记的蛋白被转移时，用抗腺病毒2型病毒粒子或抗73000 DNA结合蛋白和125i标记的金黄色葡萄球菌蛋白A序贯处理重氮甲氧基甲基纸，可以检测到低水平的病毒编码蛋白。检测到7个病毒粒子蛋白为突出带，其他几个为较弱带。通过二维凝胶转移/免疫放射自显像，检测到几种额外的病毒蛋白。利用抗dna结合蛋白血清检测Mr 73,000蛋白和Mr 41,000-48,000亚种。可以在细胞提取物中鉴定出以总蛋白质的约100,000分之一的浓度存在的蛋白质。该方法可适用于需要解决和检测少量特定蛋白质的各种生物学问题，这些蛋白质可以通过免疫识别或通过与特定放射性标记的DNA或RNA序列或激素结合来检测。
We describe a method by which complex protein mixtures are fractionated by standard one-dimensional Na-DodSO4/polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrophoresis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]-methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins could be detected by sequential treatment of diazobenzyloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 125I-labeled Staphyloccus aureus protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody, By one-dimensional gel transfer/immunoautoradiography, seven virion proteins were detected as prominent bands and several others as weaker bands. By two-dimensional gel transfer/immunoautoradiography, several additional viral proteins were detected. By use of anti-DNA binding protein serum, the Mr 73,000 protein and Mr 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.