Quantitative femto- to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis.
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.
Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA. jcoorsse@ucalgary.ca
Jens R
JR
Coorssen
Paul S
PS
Blank
Fernando
F
Albertorio
Ludmila
L
Bezrukov
Irina
I
Kolosova
Peter S
PS
Backlund
Joshua
J
Zimmerberg
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000378:metabolism; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D012617:Sea Urchins; D022142:Secretory Vesicles / Q000737:chemistry*; D013572:Synaptic Vesicles / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D002118:Calcium