Quantitative femto- to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis

Anal Biochem. 2002 Aug 1;307(1):54-62. doi: 10.1016/s0003-2697(02)00015-5.


Although immunoblotting (Western blotting) is widely used for the detection of specific proteins, it is often thought to be an inadequate technique for accurate and precise measurements of protein concentration. However, an accurate and precise technique is essential for quantitative testing of hypotheses, and thus for the analysis and understanding of proposed molecular mechanisms. The analysis of Ca(2+)-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents, requires such an unambiguous identification and a quantitative assessment of the membrane surface density of specific molecules. Newly refined immunoblotting and analysis approaches permit a quantitative analysis of the SNARE protein complement (VAMP, SNAP-25, and syntaxin) of functional secretory vesicles. The method illustrates the feasibility of the routine quantification of femtomole to attomole amounts of known proteins by immunoblotting. The results indicate that sea urchin egg secretory vesicles and synaptic vesicles have markedly similar SNARE densities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Calcium / pharmacology
  • Electrophoresis, Polyacrylamide Gel
  • Exocytosis / drug effects
  • Exocytosis / physiology*
  • Membrane Proteins / analysis*
  • Membrane Proteins / metabolism
  • Nerve Tissue Proteins / analysis*
  • Nerve Tissue Proteins / metabolism
  • Qa-SNARE Proteins
  • R-SNARE Proteins
  • SNARE Proteins
  • Sea Urchins
  • Secretory Vesicles / chemistry*
  • Synaptic Vesicles / chemistry
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins*


  • Membrane Proteins
  • Nerve Tissue Proteins
  • Qa-SNARE Proteins
  • R-SNARE Proteins
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins
  • Calcium