摘要
旨在探究Western blot检测低丰度蛋白表达及试验过程对检测结果的影响。本试验以6~8周龄C57/BL6小鼠背肩甲棕色脂肪组织(Brown adipose tissue,BAT)为试验材料,以低丰度表达的黑皮质素4型受体(Melanocortin-4-receptor,MC4R)为检测蛋白,不同蛋白处理方法及电转膜液的组分、pH为试验对照条件,探究蛋白提取方法、电转液的组分与pH对转膜效率的影响。结果显示,转膜初始电压及功率与转膜液中十二烷基磺酸钠(SDS)含量及pH极显著相关,恒流转膜时,转膜液中SDS含量与转膜电压及功率呈反比,pH为8.0时的转膜电压及功率极显著高于pH 7.6(P<0.01)和8.6的转膜电压及功率(P<0.001);MC4R及相关蛋白在醇类物混合裂解液(20%Methanol+1%Triton+1%SDS+RIPA)的蛋白处理组的表达显著优于单独RIPA的蛋白处理组(P<0.05),极显著优于Trizol的蛋白处理组(P<0.01)。综上,于MC4R蛋白而言,在裂解液的基础上添加增强剂(Triton和SDS),可促进蛋白提取的效率,提高抗体检测的灵敏性。上述结果有助于相关操作人员对主要细节的认识,提高试验的灵敏及重复性。
The main objective of this study was focused on expression of low abundance protein and details of the experimental procedure detected by Western blot.Brown adipose tissue（BAT）from 6-8 week old C57/BL6 mice were chosen as experimental materials and detected for the expression of Melanocortin-4-receptor（MC4 R）using different parameters including pH and content of Sodium Dodecyl Sulfate（SDS）in electrotransfer buffers.The effects of protein processing methods,components and pH of electrotransfer buffers on transfer efficiency were investigated.The results showed that initial voltage and power were significantly related to the content of sodium SDS and pH of the electrotransfer buffers.The concentration of SDS in the transmembrane buffer was inverse to the transfer voltage and power under the constant circulating membrane.The transmembrane voltage and power of buffer in pH 8.0 were significantly higher than those of buffers in pH 7.6（P〈0.01）and pH 8.6（P〈0.001）.The expression of relating proteins,espe-cially MC4 R,in group of alcohols mixed lysate（20%Methanol＋1%Triton＋1%SDS＋RIPA）was significantly higher than that in the single RIPA group（P〈0.05）and Trizol group（P〈0.01）.The result indicate that adding enhancers（Triton and SDS）to the lysate buffer can promote the efficiency of protein extraction and improve the sensitivity of antibody for proteins.Those procedure may help to understand the details and improve the sensitivity and repeatability of the Western blot.
作者
范奎奎
李强
杨文雅
潘登
李海军
杜晨光
FAN Kui-kui;LI Qiang;YANG Wen-ya;PAN Deng;LI Hai-jun;DU Chen-guang(College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;Vocational and Technical College of Inner Mongolia Agricultural University, Baotou 014109, China)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2018年第4期841-846,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金(31660701
31360283)
作者简介
范奎奎（1994-），男，内蒙古巴彦淖尔人，硕士生，主要从事动物组织胚胎与发育生物学研究，E-mail：fankkimau@163．com;通信作者：杜晨光，博士，教授，E—mail：duc@imau．edu．cn
