Purification and Fractionation of Insulin Secretory Granules

The iISGs and mISGs were purified following the previous protocol with minor modifications [33 (link),82 (link)]: Briefly, MIN6 cells were treated with HG for 1 h, and then homogenized in homogenization buffer [0.3 mol/L sucrose (Sigma, SLCH3216), 1 mmol/L EDTA (Merck, 654,833), 1 mmol/L MgSO₄ (Macron avantor, 6066–04), 10 mmol/L MES-KOH (Sigma, M8250), 1 mmol/L PMSF (Thermo Scientific, 36,978), pH 6.5]. The nuclear debris was removed by centrifugation at 1000 g for 5 min. The supernatant was loaded on top of a discontinuous Optiprep (Sigma, D15561) gradient, prepared by 5 layers of 30%, 23.4%, 17.6%, 13.2%, and 8.8% of Optiprep. The Optiprep gradient was centrifuged for 75 min at 100,000 g in an SW41 rotor (Beckman), and the gradients were divided into twelve fractions. Both fraction 6 (interface between 13.2% and 17.6%) and fraction 8 (interface between 17.6% and 23.4%) containing insulin were iISGs and mISGs, respectively.

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Wu S.Y., Wu H.T., Wang Y.C., Chang C.J., Shan Y.S., Wu S.R., Chiu Y.C., Hsu C.L., Juan H.F., Lan K.Y., Chu C.W., Lee Y.R., Lan S.H, & Liu H.S. (2022). Secretory autophagy promotes RAB37-mediated insulin secretion under glucose stimulation both in vitro and in vivo. Autophagy, 19(4), 1239-1257.

Publication 2022

sucrose EDTA MES-KOH PMSF Optiprep SW41 rotor

Buffer Cells Centrifugation Edta Insulin Mgso4 Sucrose

Corresponding Organization : Kaohsiung Medical University

Other organizations : Taipei Medical University, National Taiwan University

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Variable analysis

independent variables

Treatment of MIN6 cells with High Glucose (HG) for 1 h

dependent variables

Purification of iISGs and mISGs from MIN6 cells

control variables

Homogenization buffer composition (0.3 mol/L sucrose, 1 mmol/L EDTA, 1 mmol/L MgSO4, 10 mmol/L MES-KOH, 1 mmol/L PMSF, pH 6.5)
Centrifugation conditions (1000 g for 5 min to remove nuclear debris)
Optiprep gradient composition (5 layers of 30%, 23.4%, 17.6%, 13.2%, and 8.8% Optiprep)
Centrifugation conditions for Optiprep gradient (100,000 g for 75 min in an SW41 rotor)

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