Western blot鉴定背肩胛棕色脂肪黑皮质素4型受体的研究

doi:10.11843/j.issn.0366-6964.2018.04.023

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (4): 841-846.doi: 10.11843/j.issn.0366-6964.2018.04.023

Western blot鉴定背肩胛棕色脂肪黑皮质素4型受体的研究

范奎奎¹, 李强¹, 杨文雅¹, 潘登¹, 李海军¹, 杜晨光^1,2*

1. 内蒙古农业大学兽医学院, 呼和浩特 010018;
2. 内蒙古农业大学职业技术学院, 包头 014109

收稿日期:2017-09-04 出版日期:2018-04-23 发布日期:2018-04-19
通讯作者: 杜晨光,博士,教授,E-mail:duc@imau.edu.cn
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。
基金资助:
国家自然科学基金（31660701；31360283）

Identification of the Melanocortin-4-Receptor in Back Shoulder Brown Adipose Tissue by Western Blot

FAN Kui-kui¹, LI Qiang¹, YANG Wen-ya¹, PAN Deng¹, LI Hai-jun¹, DU Chen-guang^1,2*

1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Vocational and Technical College of Inner Mongolia Agricultural University, Baotou 014109, China

Received:2017-09-04 Online:2018-04-23 Published:2018-04-19

摘要/Abstract

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。

关键词: 蛋白印迹, 背肩胛骨棕色脂肪组织, 转膜液, 黑皮质素-4型受体

Abstract:

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 (MC4R) 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, especially MC4R, 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.

Key words: Western blot, back shoulder brown adipose tissue, electrotransfer buffer, Melanocortin-4-receptor

中图分类号:

Q503

范奎奎, 李强, 杨文雅, 潘登, 李海军, 杜晨光. Western blot鉴定背肩胛棕色脂肪黑皮质素4型受体的研究[J]. 畜牧兽医学报, 2018, 49(4): 841-846.

FAN Kui-kui, LI Qiang, YANG Wen-ya, PAN Deng, LI Hai-jun, DU Chen-guang. Identification of the Melanocortin-4-Receptor in Back Shoulder Brown Adipose Tissue by Western Blot[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 841-846.

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Western blot鉴定背肩胛棕色脂肪黑皮质素4型受体的研究

Identification of the Melanocortin-4-Receptor in Back Shoulder Brown Adipose Tissue by Western Blot

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