兔椎间盘软骨终板总蛋白提取方法的改良

目的:探讨快速有效的椎间盘软骨终板组织总蛋白提取方法.方法:采用一般法、外加超声法及液氮研磨法提取兔椎间盘软骨终板组织的总蛋白,并用BCA法测蛋白浓度,再用蛋白印迹检测软骨终板组织中目的蛋白1(65 kDa)、目的蛋白2(30 kDa)及β-actin(42 kDa)的表达.结果:提取107个兔椎间盘软骨终板组织,其中一般法17个,其浓度为(0.911±0.211)mg/mL；外加超声法14个,其浓度为(2.021±0.258)mg/mL；液氮研磨法76个,其浓度为(3.121±0.248) mg/mL,经统计分析,任何两种方法间蛋白浓度差异有统计学意义,液氮研磨法所得蛋白浓度最高;且液氮研磨法所得的总蛋白经蛋白印迹得到目的蛋白-1、目的蛋白-2及β-actin的清晰条带.结论:液氮研磨法可以作为少量软骨组织蛋白提取的一种理想方法,可以在一般实验室中展开,进而可在蛋白质水平上进行相关功能的研究.

Methods improvement for protein extraction from cartilage endplate in rabbit intervertebral disc

Objective To establish a quick and efficient method for total protein extraction from cartilage endplate in rabbit intervertebral disc.Methods Total protein was extracted respectively by the following methods,including general method,general method plus ultrasound,liquidnitrogen-grinding.Protein concentration was determined by BCA dye-binding assay and used for target protein-1(65 kDa),target protein-2(30 kDa) and β-actin(42 kDa) expression assay by Western blot.Results One hundred and seven cartilage endplate samples were used in this study.The general method was used for protein extraction from 17 samples,and the protein concentration was(0.911 ± 0.211)mg/mL.The general method plus ultrasound was used for protein extraction from 14 samples,and the protein concentration was(2.021 ± 0.258)mg/mL.The liquidnitrogen-grinding method was used for protein extraction from 76 samples,the protein concentration was(3.121 ± 0.248)mg/mL.The statistical analysis revealed that there was a statistical difference in protein concentration between any two groups with different methods.The highest protein extraction was prepared by using the liquidnitrogen-grinding method,and the distinct bands of target protein-1,target protein-2 and β-actin could be shown by Western blot assay.Conclusion Liquidnitrogen-grinding is an efficient and easy method for protein extraction from the cartilage tissue for further protein function-related study.