30C-12 |
Enzyme linked immunosorbent assay (ELISA) for sulfur-rich protein (SRP) in soybeans (Glycine max L.) |
E. MONAGHAN1, M. Venkatachalam1, S. S. Teuber2, K. H. Roux3, and S. K. Sathe1. (1) Department of Nutrition, Food and Exercise Sciences, Florida State University, 402 Sandels Bldg., College of Human Sciences, Tallahassee, FL 32306-1493, (2) Division of Rheumatology, Allergy and Clinical Immunology, University of California, Davis, CA 95616, (3) Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370 The sulfur-rich protein (SRP) in soybeans is a 7S basic globulin that accounts for up to 5% of the total extractable seed proteins. Sulfur-containing amino acids are the limiting amino acids in most food legumes and therefore there is a continued interest in increasing the expression of sulfur containing proteins in legumes to improve protein quality. The objective of our investigation was to develop a sensitive method for detection of SRP and use the method to detect the presence of SRP or SRP-like proteins in various plant seeds. Rabbit polyclonal antibodies (pAbs) raised against SRP were used in inhibition ELISA and Western blotting to develop a sensitive assay and to assess cross-reactivity of pAbs to non soybean proteins. Typically, primary and secondary antibody dilutions of 104 and 5 x 103 in 0.1% BSA in borate saline buffer (BSB, 0.1M, pH 8.45), respectively, were used. All protein extractions were done in BSB and cross reactivity was assessed by comparing sample IC50 (50% inhibitory concentration) to IC50 value for SRP. Inhibition ELISA assays could detect SRP at concentrations as low as 300 ng/ml in aqueous extracts. Inhibition ELISA and Western blotting assays indicated the presence of cross-reactive proteins in several dry beans including Tepary, Great Northern, Hyacinth, Winged, Small red and Black eye peas as well as wheat bran and wheat berries. These results indicate presence of sulfur rich proteins in legumes other than soybeans. Increased expression of such sulfur rich proteins has the potential to improve legume protein quality due to improved balance of essential amino acids.
Session 30C, Food Chemistry: Proteins
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