Measuring relative insulin secretion using a co-secreted luciferase surrogate

Research output: Contribution to journalArticle

Abstract

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic β cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

Original languageEnglish (US)
Article numbere59926
JournalJournal of Visualized Experiments
Volume2019
Issue number148
DOIs
StatePublished - Jun 1 2019

Fingerprint

Insulin
Luciferases
Assays
C-Peptide
Antibodies
Peptides
Secretory Vesicles
Luminescence
Costs and Cost Analysis
Gages

Keywords

  • Biology
  • Diazoxide paradigm
  • Gaussia
  • Insulin
  • Issue 148
  • Pancreatic beta cell
  • Secreted luciferase
  • Secretion assay

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Measuring relative insulin secretion using a co-secreted luciferase surrogate. / Kalwat, Michael; Cobb, Melanie H.

In: Journal of Visualized Experiments, Vol. 2019, No. 148, e59926, 01.06.2019.

Research output: Contribution to journalArticle

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