Quantification of green fluorescent protein-(GFP-) tagged membrane proteins by capillary gel electrophoresis

Analyst. 2017 Oct 7;142(19):3648-3655. doi: 10.1039/c7an00981j. Epub 2017 Aug 31.

Abstract

A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A2A receptor (A2AAR), and connexin 43 (Cx43). Quantification of SV2A and A2AAR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

MeSH terms

  • Animals
  • CHO Cells
  • Connexin 43 / analysis
  • Cricetulus
  • Electrophoresis, Capillary*
  • Green Fluorescent Proteins / analysis*
  • Humans
  • Lasers
  • Membrane Glycoproteins / analysis
  • Membrane Proteins / analysis*
  • Mice
  • Nerve Tissue Proteins / analysis
  • Receptor, Adenosine A2A / analysis

Substances

  • ADORA2A protein, human
  • Connexin 43
  • Membrane Glycoproteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Receptor, Adenosine A2A
  • Green Fluorescent Proteins
  • SV2A protein, human