An optimization technique to purify the pre-ribosome and ribosome from mammalian cells using continuous sucrose density gradient ultracentrifugation
10.3760/cma.j.issn.1673-4181.2015.05.002
- VernacularTitle:一种连续蔗糖密度梯度离心分离哺乳动物细胞核糖体前体和核糖体的技术优化
- Author:
Shuangshuang LIANG
;
Meichao JI
;
Xiaoqing HU
;
Chenghua FU
;
Changiun ZHU
;
Zhixiong DONG
- Publication Type:Journal Article
- Keywords:
Continuous sucrose density gradient;
Ribosome;
Pre-ribosome;
RPL15
- From:
International Journal of Biomedical Engineering
2015;38(5):262-265,281
- CountryChina
- Language:Chinese
-
Abstract:
Objective To purify pre-ribosome and ribosome of mammalian ceils using continuous sucrose density gradient ultracentrifugation.Methods Continuous sucrose density gradient was established by ultracentrifugation,and the continuous sucrose density gradient of 10％-30％ and 10％-45％ were used to extract the pre-ribosome and ribosome in mammalian cells,respectively.The mammalian cell lysis buffer was added to the established continuous sucrose density gradient.Pre-ribosome and ribosome with different sedimentation coefficients were collected and the A260 absorbance of each sample was measured.Proteins of each sample were extracted to detect the large subunit protein,RPL15 by Western Blot.Results Large subunit ribosomal protein RPL15 exists on 60S of the pre-ribosome,and also on 60S,80S and polyribosome of mature ribosome.Conclusions The continuous sucrose density gradient,which is established by the swing-out rotor,can be used to isolate the pre-ribosome and ribosome of mammalian cells rapidly.This method has the advantages of good separation effect and simple operation,which provides a good method for rapid and large amount preparation and separation of various kinds of ribosomes.
