Chemiluminescence detection of proteins from single cells

Peter G. Gillespie, A. J. Hudspeth

Research output: Contribution to journalArticle

100 Scopus citations

Abstract

The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro{1,2dioxetane-3,2′-tricyclo-[3.3.1.1 3.7]decan}-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, ehemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting ≈1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.

Original languageEnglish (US)
Pages (from-to)2563-2567
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number6
DOIs
StatePublished - Jan 1 1991

Keywords

  • Avidin
  • Biotin
  • Immunoblot
  • Photoreceptor

ASJC Scopus subject areas

  • General

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