An improved enzyme-linked immunoabsorbent assay protocol for the detection of small lytic peptides in transgenic grapevines (Vitis vinifera) |
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Authors: | Zhijian Li Subramanian Jayasankar D J Gray |
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Institution: | (1) Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2725 Binion Road, 32703-8504 Apopka, FL, USA |
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Abstract: | An enzyme-linked immunoabsorbent assay (ELISA) protocol was developed for the detection of small lytic peptides in transgenic
grapevines (V. vinifera). The protocol requires a high concentration of protease inhibitor in the extraction buffer; the use of antiserum cross-absorbed
with control tissue, an increased concentration of blocking reagents in the antiserum buffer, and performing all coating and/or
binding processes at 37°C while reducing the time period for each step to 1 h. The procedure greatly reduced protein degradation,
increased the signal-to-noise ratio, and it allowed the effective detection of the Shiva-1 lytic peptide (5 kDa) at concentrations
as low as 0.1 μM. This procedure made it possible for routine analysis of transgene expression in Shiva-1 gene-containing
transgenic grape plants. |
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Keywords: | antimicrobial proteins immunoassay protein detection transgene expression transgenic grape |
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