PRAS40(Ser183)磷酸化多克隆抗体的制备及检测

Preparation and detection of phosphorylated PRAS40 (Ser183) polyclonal antibody

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 （proline-rich Akt substrate 40 kD） associates with mammalian target of rapamycin complex I（mTORC1）, serine 183 site （Ser183） of PRAS40 can be phosphorylated by mTORC1. To prepare the phosphorylated PRAS40 （Ser183） antibody, We chosen 10-amino acid including Ser183 as antigen peptide through antigenicity and hydrophobicity analysis, hinged on keyhole limpet hemocyanin （KLH）, and used the KLH-peptide to immunize rabbits. After antibody serum titer detection by enzyme linked immunosorbent assay （ELISA）, the antibody was purified with rProtein A sepharose fast flow and dephosphorylated antigen membrane. The antibody titrate reached 1：10 000 after purification and its special property was enhanced with absorption treatment of dephosphorylated antigen membrane. In addition, we used rabbit anti-PRAS40 antibody and the phosphorylated PRAS40 （Ser183） antibody to detect PRAS40 expression in several cell lines, including the normal cells HL7702, HEK293, tumor cells HepG2, A549 and S180. There were no quite difference among these cells; otherwise, we observed the decreased phosphorylation level of Ser183 after amino acid withdrawal treatment. Therefore, the polyclonal phosphorylated PRAS40 （Ser183） antibody was specific to PRAS40 （Ser183） site and could be used for the function study of PRAS40.