Abstract
The suitability of the Bradford protein assay for measuring plant protein was evaluated and a standard method developed. The assay involves extraction of dried, fresh, or frozen plant material in 0.1 NaOH for 30 min. Replicate 100-μl aliquots of centrifuged supernatant are assayed with 5 ml Bio-Rad Bradford dye reagent (Coomassie brilliant blue G-250) diluted 1:4 and containing 3 mg/ml soluble polyvinylpyrollidone. Absorbance at 595 nm is recorded after 15 min against an NaOH blank. Samples are calibrated against a ribulose 1,5-diphosphate carboxylase-oxygenase standard in NaOH. Procedures for plant preparation, extraction stability, the effects of phenol removal and quinone formation, and assay recovery are evaluated. Assay absorbance stability and techniques for increasing absorbance stability are reported. Changes in protein quality are briefly discussed.
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References
Bensadoun, A., andWeinstein, D. 1976. Assay of proteins in the presence of interfering materials.Anal. Biochem. 70:241–250.
Bio-Rad Laboratories 1985. Bio-Rad Protein Assay. Publication 85-0192, Bio-Rad Laboratories, Richmond, California.
Bradford, M.M. 1976. A rapid and sensitive method for the estimation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248–254.
Compton, S.J., andJones, G.G. 1985. Mechanism of dye response and interference in the Bradford protein assay.Anal. Biochem. 151:369–374.
Denno, R.F., Douglass, L.W., andJacobs, D. 1986. Effects of crowding and host plant nutrition on a wing-dimorphic planthopper.Ecology 67:116–123.
Ellis, R.J. 1977. Protein synthesis by isolated chloroplasts.Biochim. Biophys. Acta 463:185–216.
Eze, J.M.O., andDumbroff, E.B. 1982. A comparison of the Bradford and Lowry methods for the analysis of protein in chlorophyllous tissue.Can.J. Bot. 60:1046–1049.
Faeth, S.H. 1985. Quantitative defense theory and patterns of feeding by oak insects.Oecologia 68:34–40.
Gnaiger, E., andBitterlich, G. 1984. Proximate biochemical composition and caloric content calculated from elemental CHN analysis: A stoichiometric concept.Oecologia 62:289–298.
Harborne, J.B. 1973. Phytochemical Methods. Chapman and Hall, London.
Hare, J.D. 1983. Seasonal variation in plant-insect associations. Survival ofLeptinotarsa decemlineata onSolatium dulcamara.Ecology 64:345–361.
Krystal, G., Macdonald, C., Munt, B., andAshwell, S. 1985. A method for quantitating nanogram amounts of soluble protein using the principle of silver binding.Anal. Biochem. 148:451–460.
Loomis, W.D. 1969. Removal of phenolic compounds during the isolation of plant enzymes, pp. 555–563,in J.M. Lowenstein (ed.). Methods in Enzymology, Vol. 13. Academic Press, New York.
Loomis, W.D., andBattaile, J. 1966. Plant phenolic compounds and the isolation of plant enzymes.Phytochemistry 5:423–438.
Loomis, W.D., Lile, J.D., Sandstrom, R.P., andBurbott, A.J. 1979. Adsorbent polystyrene as an aid in plant enzyme isolation.Phytochemistry 18:1049–1054.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., andRandall, R.J. 1951. Protein measurement with the Polin phenol reagent.J. Biol. Chem. 193:265–275.
Martin, M.M., Rockholm, D.C., andMartin, J.S. 1985. Effects of surfactants, pH, and certain cations of precipitation of proteins by tannins.J. Chem. Ecol. 11:485–494.
Marks, D.L., Buchsbaum, R., andSwain, T. 1985. Measurement of total protein in plant samples in the presence of tannins.Anal. Biochem. 147:136–143.
Mattoo, R.L., Ishaq, M., andSaleemuddin, M. 1987. Protein assay by Coomassie brilliant blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases.Anal. Biochem. 163:376–384.
Mattson, W.J. 1980. Herbivory in relation to plant nitrogen content.Annu. Rev. Ecol. Syst. 11:119–161.
Maynard, A.B., andLoosli, J.K. 1969. Animal Nutrition. McGraw-Hill, New York.
Peterson, G.L. 1983. Determination of total protein, pp. 95–119,in C.W. Hirs and S. N. Timasheffs (eds.). Methods in Enzymology, Vol. 91. Academic Press, New York.
Pierpont, W.A. 1969a. o-Quinones formed in plant extracts: Their reactions with amino acids and peptides.Biochem J. 112:609–616.
Pierpont, W.A. 1969b. o-Quinones formed in plant extracts: Their reaction with bovine serum albumin.Biochem. J. 112:619–627.
Riegler, E. 1914. Eine metrischer Bestimmungsmethode bei Weisses.Z. Anal. Chem. 53:242–245.
Scriber, J.M., andSlansky, F., Jr. 1981. The nutritional ecology of immature insects.Annu. Rev. Entomol. 26:183–211.
Slansky, F., Jr., andScriber, J.M. 1985. Food consumption and utilization, pp. 87–163,in G. A. Kerkut and L.I. Gilbert (eds.). Comprehensive Insect Biochemistry and Physiology, Vol. 4. Pergamon Press, Oxford.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., andKlenk, D.C. 1985. Measurement of protein using bicinchoninic acid.Anal. Biochem. 150:76–85.
Trumble, J.T., Hare, J.D., Musselman, R.C., andMcCool, P.M. 1987. Ozone-induced changes in host-plant suitability: Interactions ofKiefena lycopersicella andLycopersicon esculentum.J. Chem. Ecol. 13:203–217.
Warburg, O., andChristian, W. 1941/1942. Isolierung und Kristallisation des garungsferments Enolase.Biochem. Z. 310:384–421.
Wehr, H.M., andLoomis, W.D. 1971. Amino acid modifications and digestibility in tanned protein.Plant Physiol. 47(Suppl.):281.
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Jones, C.G., Daniel Hare, J. & Compton, S.J. Measuring plant protein with the Bradford assay. J Chem Ecol 15, 979–992 (1989). https://doi.org/10.1007/BF01015193
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DOI: https://doi.org/10.1007/BF01015193