Islet Protein Extraction and Western Blotting

Hand-picked islets were washed once with PBS and then resuspended in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors cocktails (Sigma) as described^{32 (link)}. Islets were lysed by repeated pipetting or triturating through a 30G syringe needle. Lysates were resolved on a 4–12% NuPAGE Bis-Tris gel, and then electrotransferred to nitrocellulose membrane for western blotting. Antibodies used were mouse anti-proinsulin (ALPCO), guinea pig anti-insulin (Covance), and mouse anti-vinculin (Millipore). Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc., with protein visualized by ECL (Millipore).

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Mao Y., Schoenborn J., Wang Z., Chen X., Matson K., Mohan R., Zhang S., Tang X., Arunagiri A., Arvan P, & Tang X. (2022). Transgenic overexpression of microRNA-30d in pancreatic beta-cells progressively regulates beta-cell function and identity. Scientific Reports, 12, 11969.

Publication 2022

protease inhibitors and phosphatase inhibitors cocktails guinea pig anti-insulin mouse anti-vinculin Horseradish peroxidase-conjugated secondary antibodies ECL

Antibodies Bis tris Buffer Guinea pig Horseradish peroxidase Inhibitors Insulin Membrane Mouse Needle Nitrocellulose Phosphatase Proinsulin Protease inhibitors Protein Ripa Syringe Vinculin

Corresponding Organization :

Other organizations : Michigan Technological University, University of Michigan–Ann Arbor

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Variable analysis

independent variables

Treatment of islets with RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors cocktails

dependent variables

Protein levels of proinsulin
Protein levels of insulin
Protein levels of vinculin

control variables

Washing of islets with PBS
Lysing of islets by repeated pipetting or triturating through a 30G syringe needle

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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