A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins.
Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78ng/ml to 77pg/ml (or 74-0.1fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.
Geumann
Constanze
C
Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. cgeumann@sysy.com <cgeumann@sysy.com>
Grønborg
Mads
M
Hellwig
Michaela
M
Martens
Henrik
H
Jahn
Reinhard
R
D000818:Animals; D002138:Calibration; D003902:Detergents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D012995:Solubility; D013570:Synaptic Membranes / Q000737:chemistry*
D003902:Detergents; D008565:Membrane Proteins; D011994:Recombinant Proteins