An improved enzyme-linked immunoabsorbent assay protocol for the detection of small lytic peptides in transgenic grapevines (Vitis vinifera)
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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.
Key wordsantimicrobial proteins immunoassay protein detection transgene expression transgenic grape
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- Arce P, Moreno M, Gutierrez M, Gebauer M, Dell’Orto P, Torres H, Acuna I, Oliger P, Venegas A, Jordana X, Kalazich J, and Holuigue L (1999) Enhanced resistance to bacterial infection byErwinia carotovora subsp.atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes. Amer J Potato Res 76: 169–177.CrossRefGoogle Scholar
- Crowther JR (1995) Methods in Molecular Biology, Vol. 42-ELISA: Theory and Practice. Humana Press, Totowa, NJ.Google Scholar
- Jaynes JM (1993) Use of genes encoding novel lytic peptides and proteins that enhance microbial disease resistance in plants. Acta Hort 336: 33–39.Google Scholar
- Sambrook J, Fritsch E, and Maniatis T (1989) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New York, USA.Google Scholar
- Tijssen P (1985) Practice and theory of enzyme immunoassay. Elsevier Sci Publishers BV, New York, pp. 344–350.Google Scholar