Problems associated with determining protein concentration
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Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric analysis and densitometry of stained protein gels, are applied, but perhaps to a lesser extent. However, the reliability of all of the above techniques is questionable and dependent to some extent on the protein to be assayed. In this paper we describe problems we encountered when using some of the foregoing techniques to quantify the concentration of poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1), a nuclear enzyme found in most eukaryotes. We also describe how, by using a fluorescence-based assay and amino acid analysis, we overcame the problems we encountered.
- Burkle, A. (2001) Physiology and pathophysiology of poly(ADP-ribosyl)ation. BioEssays, 23, 795–806. CrossRef
- Smith, S. (2001) The world according to PARP. TIBS, 26, 174–179.
- Knight, M.I. and Chambers, P.J. (2001) Production, extraction and purification of human poly(ADP-ribose) polymerase-1 (PARP-1) with high specific activity. Prot. Exp. Purific., 23, 453–458. CrossRef
- Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with folin phenol reagent. J. Biol. Chem., 103, 265–275.
- Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1996) “Quantitation of Proteins” in Current Protocols in Molecular Biology, John Wiley and Sons Inc., USA.
- Markwell, M.A., Kass, S.M., Tolbert, N.E. and Bieber, L.L. (1981) Protein determination in membrane and lipoprotein samples. Meths. Enzymol. 72, 296–303. CrossRef
- Hammond, B.W. and Kruger, N.J. (1988) “The Bradford Method for Protein Quantitation,” in New Protein Techniques, edited by Walker, J.M., Humana Press, Totowa, N.J., USA.
- Bradford, M.M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 131, 248–254. CrossRef
- Compton, S.J. and Jones, C.G. (1985) Mechanism of dye response and interference in the Bradford protein assay. Anal. Biochem. 151, 369–374. CrossRef
- Stoscheck, C.M. (1990) Quantitation of Protein. In Guide to Protein Purification, Deutscher, M. P., ed., Academic Press, San Diego, California.
- Promega (1993) Promega Protein Guide: Tips and Techniques, prepared and edited by Promega Corporation, Madison, WI, USA.
- Patel, K., Easty, D.J. and Dunn, M.J. (1988) Detection of Proteins in Polyacrylamide gels using an ultrasensitive silver staining technique. In New Protein Techniques, Walker, J.M., ed., Humana Press, Totowa, N.J., USA.
- Rabilloud, T. (1992) A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis, 13, 429–439. CrossRef
- Molecular Probes (2000) NanoOrange® Protein Quantitation Kit Manual, prepared and edited by molecular Probes, Eugene, OR, USA.
- Jacobs, D.I., van der Heijden, R. and Verpoorte, R. (2000) Proteomics in plant biotechnology and secondary metabolism research. Phytochemical Anal. 11, 277–287. CrossRef
- LKB Pharmacia (1986) Laboratory Manual, LKB 2050, Midget Electrophoresis Unit and LKB 2051, Midget MultiBlot Electrophoretic Transfer Unit. LKB Produckter AB, Sweden, pp 26–28.
- Biorad (1998) SYPRO® Orange Protein Gel Stain Instruction Manual, prepared and edited by Biorad Laboratories, Hercules, CA, USA.
- Steinberg, T.H., Jones, L.J., Haugland, R.P., and Singer, V.L. (1996) Sypro orange and sypro red protein gel stains: one-step fluorescent staining of denturing gels for the detection of nanogram level of protein. Anal. Biochem. 239, 223–237. CrossRef
- Problems associated with determining protein concentration
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