title: An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells.
abstract: Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from thetrans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.|University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.|University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Xia
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.|University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Lifen
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.|University of Chinese Academy of Sciences, Beijing, 100049 China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.
authors.forename: Pingsheng
authors.initials: P
authors.lastname: Liu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.
authors.forename: Eli
authors.initials: E
authors.lastname: Song
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China.
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
keywords: Density gradient; Immature insulin secretary granules (iISGs); Insulin; Mature insulin secretary granules (mISGs); Subcellular fractionation