ECM Enrichment of Islet Cells

Normal, hyperplastic and angiogenic islets and insulinomas were homogenized with a Bullet Blender (Next Advance, Averill Park, NY) according to the manufacturer’s instructions. Decellularization (removal of intracellular proteins) and concomitant enrichment of ECM proteins were performed using the CNMCS compartmental protein extraction kit (Millipore, Billerica, MA) as previously described8 (link)32 . The effectiveness of the ECM enrichment was monitored by immunoblotting using the following primary antibodies: rabbit anti-collagen I, mouse anti-GAPDH and rabbit anti-histones (Millipore, Billerica, MA), the rabbit anti-actin antibody (serum 14–1) was generated in our laboratory.

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Naba A., Clauser K.R., Mani D.R., Carr S.A, & Hynes R.O. (2017). Quantitative proteomic profiling of the extracellular matrix of pancreatic islets during the angiogenic switch and insulinoma progression. Scientific Reports, 7, 40495.

Publication 2017

Bullet Blender CNMCS compartmental protein extraction kit rabbit anti-collagen I mouse anti-GAPDH rabbit anti-histones

Actin Angiogenic Anti antibody Antibodies Collagen i Ecm proteins Gapdh Histones Hyperplastic Insulinomas Intracellular Mouse Protein Rabbit Serum

Corresponding Organization : Howard Hughes Medical Institute

Other organizations : Broad Institute

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Variable analysis

independent variables

Homogenization method (Bullet Blender)
Decellularization and ECM enrichment method (CNMCS compartmental protein extraction kit)

dependent variables

Enrichment of ECM proteins

control variables

Sample types (normal, hyperplastic and angiogenic islets, and insulinomas)

controls

Positive control: Immunoblotting using primary antibodies against collagen I, GAPDH, and histones
Negative control: Not explicitly mentioned

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