High Sensitive Quantitative Binding Assays Using a Nanoluciferase-Fused Probe for Analysis of ALG-2-Interacting Proteins

Methods Mol Biol. 2019:1929:501-516. doi: 10.1007/978-1-4939-9030-6_31.

Abstract

Many non-catalytic cellular proteins exert biological functions by formation of stable or transient complexes with other proteins. Analysis of the signal-induced physical interactions is important to understand their physiological roles in cells. Here we describe a biochemical method for assessing the binding of ALG-2 (gene name, PDCD6) to its target proteins that are immunoprecipitated from cell lysates. Application of nanoluciferase (Nluc)-fused ALG-2 enables a rapid quantitative evaluation of Ca2+-dependent interactions of target proteins with ALG-2 in vitro binding assays.

Keywords: ALG-2; Calcium-binding protein; EF-hand; In vitro binding; NanoLuc; Protein–protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / genetics*
  • Apoptosis Regulatory Proteins / metabolism*
  • Calcium / metabolism*
  • Calcium-Binding Proteins / genetics*
  • Calcium-Binding Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • Luciferases / genetics*
  • Nanotechnology
  • Protein Binding
  • Recombinant Fusion Proteins / metabolism

Substances

  • Apoptosis Regulatory Proteins
  • Calcium-Binding Proteins
  • PDCD6 protein, human
  • Recombinant Fusion Proteins
  • Luciferases
  • Calcium