低浓度多聚甲醛固定对蛋白质免疫印迹技术的改良  

Improved Immunoblot by Brief Application of Low Dose Paraformaldehyde

作　　者：孙忠权[1] 陆文[1] 艾恒[2] 

机构地区：[1]浙江大学,杭州310012 [2]浙江医学高等专科学校,杭州310053

出　　处：《中国生物化学与分子生物学报》2013年第12期1187-1193,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省教育厅科研项目(No.Y201122469);浙江医学高等专科学校博士启动项目(No.2013B01)~~

摘　　要：蛋白质免疫印迹(protein immunoblot,或Western blot)是一种广泛应用于检测细胞或组织蛋白质表达及蛋白质翻译后修饰的方法.前期的研究发现,使用低浓度(0.4%)多聚甲醛在蛋白质免疫印迹封闭环节前做膜固定,有助于提高蛋白质的检测效果.本文通过设置多聚甲醛的不同浓度和固定时间,进一步探索其在蛋白质免疫印迹技术中的应用.结果发现,低浓度(≤0.4%)多聚甲醛处理30 min,能明显提升蛋白质的检测效率.通过检测不同大小蛋白质的固定效果发现,大分子量的蛋白质使用多聚甲醛固定效果不明显,中等分子量和小分子量的蛋白质的固定效果较佳.综上研究表明,在中等或小分子量的蛋白质免疫印迹检测中,封闭环节前加入低浓度多聚甲醛固定,可以提高蛋白质的检测效果.Protein immunoblot （Western blot） and posttranslational modification of proteins has been a widely adopted method for studying expression in cells and tissues. Previous studies have shown that application of low concentration （ 0.4% ） of paraformaldehyde （ PFA ）, an additional step specifically inserted between transferring and blocking the membrane, can improve detection of target protiens in immunoblot. In our research, to find out the most effective concentration and time course of PFA treatment in immunoblot, a series of concentration of PFA and time course of treatment were set up in experiments for detecting various proteins. In general, it is sufficient to improve detection in immunoblot by low concentration （ ≤0.4% ） and 30 minutes of application of PFA, However, fixation of proteins of large molecule weight did not manifest any improved effect for detection, while fixation of medium and small proteins showed significant improvement. Surprisingly, fixation of exogeneous GFP, which is a 28 kD protein expressed in HEK293 ceils, displayed no significant improvement. In conclusion, our study suggests that fixation of proteins of medium and small molecule weight by PF will improve the detection efficiency.

关 键 词：蛋白质免疫印迹 多聚甲醛 固定 

分 类 号：Q-33[生物学]