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Related Experiment Videos

A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters.

R Jahn, W Schiebler, P Greengard

    Proceedings of the National Academy of Sciences of the United States of America
    |March 1, 1984
    PubMed
    Summary

    A new immunoassay quantifies synapsin I and a 36 kDa membrane protein in rat brain synaptic vesicles. This method offers accurate, sensitive, and parallel sample analysis without antigen iodination.

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    Area of Science:

    • Neuroscience
    • Biochemistry
    • Analytical Chemistry

    Background:

    • Synaptic vesicles are crucial for neurotransmission.
    • Accurate quantification of vesicle proteins like synapsin I is essential for understanding neuronal function.
    • Existing methods may have limitations in sensitivity, throughput, or sample preparation.

    Purpose of the Study:

    • To develop and validate a novel immunoassay for quantifying synapsin I (Protein I) and a 36,000-dalton membrane protein from rat brain synaptic vesicles.
    • To establish conditions for optimal assay performance, including protein retention, background reduction, and adjustable sensitivity.
    • To demonstrate the assay's utility for analyzing crude tissue samples and handling a large number of samples in parallel.

    Main Methods:

    • Samples are applied to nitrocellulose membrane filters.

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  • Sequential incubation with specific antibodies and 125I-labeled Protein A.
  • Radioactivity is measured using a gamma scintillation counter.

    • Main Results:

    • Optimized conditions prevent protein loss and quench background radioactivity.
    • The assay demonstrates linearity over a 20- to 50-fold range.
    • High sensitivity achieved: 10 pmol of synapsin I and 50 ng of total vesicle membrane protein accurately measured.

    Conclusions:

    • A robust and sensitive immunoassay for quantifying synaptic vesicle proteins has been developed.
    • The method is accurate with crude samples, requires no antigen iodination, and allows for high-throughput analysis.
    • This technique is adaptable for the measurement of various other proteins.