Crawford L V, O'Farrell P Z
J Virol. 1979 Feb;29(2):587-96. doi: 10.1128/JVI.29.2.587-596.1979.
We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.
我们通过免疫沉淀和二维凝胶电泳分析了大T抗原和小t抗原的不同种类。T抗原种类直接进行电泳，或在用N-乙基马来酰亚胺还原和烷基化后进行电泳。用N-乙基马来酰亚胺处理可提高二维凝胶电泳对大T抗原的分辨率，并且是二维凝胶上分辨小t抗原所必需的。大T抗原不会形成离散的蛋白质斑点，而是在等电聚焦凝胶上从大约pH 6.5到6.9形成一条条带。在非平衡条件下进行电泳时，小t抗原在大约pH 7.2处形成一个尖锐的蛋白质斑点，这种非平衡条件扩展了pH梯度，以包括具有碱性等电点的蛋白质。用N-乙基马来酰亚胺处理会降低十二烷基硫酸钠凝胶电泳过程中T抗原种类的迁移率。我们认为分子量的明显增加是由于N-乙基马来酰亚胺与这些蛋白质富含半胱氨酸的区域结合所致。使用不诱导小t抗原合成但产生小t相关多肽的猴病毒40的活缺失突变体，将小t抗原富含半胱氨酸的区域定位在猴病毒40遗传图谱上的0.54至0.59之间。
