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PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。
PRAS40 is a proline-rich, 40 kD Akt substrate protein that binds to rapamycin mammalian cell-targeted complex 1 (mTORC1) and its threonine 183 site (Ser183) is activated by mTORC1 phosphate The In order to prepare PRAS40 (Ser183) phosphorylated polyclonal antibody, we designed the polypeptide antigen by hydrophobic antigen analysis of protein, and used it to immunize rabbits to obtain antiserum. The titer of ELISA was 1: 10000. The result of Western blotting showed that, The antibody purified by rProtein A Sepharose affinity chromatography and adsorbed on non-phosphorylated antigenic strips could obviously increase the specificity of phosphorylation antibody. The anti-phosphorylation activity of normal cells HL7702, HEK293 and PRAS40 (Ser183) The detection of tumor cells HepG2, A549 and S180 showed that there was no significant difference in the expression of phosphorylated Ser183 in different cells, but the phosphorylation level of S183 in HEK293 cells treated with cell starvation obviously decreased with the decrease of amino acid content . Therefore, the antibody prepared in this experiment can be used to study the function of PRAS40 (Ser183) phosphorylation site.