Extraction of cytoskeletons and associated proteins using subcellular proteome fractionation technique.
- Author:
Zhen CAI
1
;
Shilong XIONG
;
Yuan ZHOU
;
Huicai JI
;
Chunyan XINAG
;
Lei ZHENG
Author Information
- Publication Type:Journal Article
- MeSH: Cytoskeleton; chemistry; Electrophoresis, Gel, Two-Dimensional; methods; Humans; Proteome; analysis; Proteomics; methods; Subcellular Fractions
- From: Journal of Southern Medical University 2013;33(5):698-702
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo develop a method for extracting cytoskeletons and cytoskeleton-associated proteins for proteomic analysis.
METHODSA subcellular sequential proteome extraction method was exploited. The extraction procedure was optimized and controlled according to observed cell morphology changes and one- and two-dimensional electrophoresis images. The extraction efficiency and selectivity were evaluated by Western blotting and mass spectrometry.
RESULTSFour extracted fractions clearly displayed distinct patterns. Western blotting detected the fraction-marker proteins FAK, integrin-β1, histone H1 and cytokeratin 19 only in their expected fractions. About 90% of the protein spots in the cytoskeleton fraction were identified by mass spectrometry as cytoskeleton and/or its associated proteins.
CONCLUSIONThe subcellular proteome sequential fractionation method facilitates the detection of proteins of low abundance and shows a high reproducibility and selectivity, and thus can serve as an ideal pre-fractionation method prior to two-dimensional electrophoresis.
