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目的:利用双向电泳技术分析大鼠肾小球蛋白质,以建立肾小球蛋白质组分析的方法。方法:选取体重约200g的雄性Wistar大鼠,分离肾小球并提取蛋白质。使用24cm、pH3-11的IPG胶条对蛋白质进行一相等电聚焦,采用12.5%聚丙烯酰胺凝胶进行二相分离。Decyder软件分析凝胶上的蛋白质点,切取1个蛋白质点,经过胰蛋白酶消化后,使用飞行质谱获取肽指纹谱,并用Mascot软件分析。结果:纯化的肾小球纯度在90%以上,在双向电泳的凝胶上,共发现1240个肾小球蛋白质;切取的蛋白质点经鉴定为CAPZA2蛋白质,Mascot得分为137分(阈值为61分)。结论:成功建立了双向电泳分析大鼠肾小球蛋白质组的方法。
OBJECTIVE: To analyze rat glomerular protein by two-dimensional electrophoresis to establish a method for the analysis of glomerular proteome. Methods: Male Wistar rats weighing about 200 g were selected to separate glomeruli and extract protein. The proteins were subjected to an equal power focusing using a 24 cm, IP3-11 pHG strip and two phase separations using a 12.5% polyacrylamide gel. Decyder software analyzed the protein spots on the gel, cut one protein spot, digested with trypsin, obtained the peptide fingerprint using flight mass spectrometry, and analyzed using Mascot software. RESULTS: Purified glomeruli were over 90% pure. A total of 1240 glomerular proteins were found on the two-dimensional gel. The cut protein spots were identified as CAPZA2 protein and Mascot score was 137 (threshold: 61 ). Conclusion: Two-dimensional gel electrophoresis was successfully established to detect rat glomerular proteome.