Abstract
The accurate quantitation of proteins and an analysis of their purity are essential in numerous areas of scientific research, and is a critical factor in many clinical applications. The large number and variety of techniques employed for this purpose is therefore not surprising. The selection of a suitable assay is dependent on such factors as the level of sensitivity required, the presence of interfering agents, and the composition of the protein itself. Here, protocols for the most commonly used protein determination methodologies are outlined, as well as for the more recently adapted technique of quantitative immuno-Polymerase Chain Reaction.
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References
Rosenburg IM (2005) Getting started with protein purification. In: Protein analysis and purification: benchtop techniques, 2nd edn. Birkhäuser, Boston, MA, pp 118–152
Lovrien R, Matulis D (2005) Assays for total protein. In: Coico R, Kowalik T, Quarles J, Stevenson B, Taylor R (eds) Current protocols in microbiology. Wiley, Hoboken, NJ, pp A.3.A.1–A.3.A.14
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Baines D (2001) Analysis of purity. In: Roe S (ed) Protein purification techniques: practical approach, 2nd edn. Oxford University Press, Oxford, pp 27–46
Noble JE, Bailey MJA (2009) Quantitation of protein. In: Burgess RR, Deutscher MP (eds) Methods in enzymology. Guide to protein purification, vol 463, 2nd edn. Academic, New York, pp 73–95
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Johnson M (2012) Protein quantitation. Mater Methods 2:115. Available via LABOME. http://www.labome.com/method/Protein-Quantitation.html. Accessed 23 Dec 2015
Olson BJSC, Markwell J (2007) Assays for determination of protein concentration. In: Coligan JE, Dunn BE, Speicher DW, Wingfield PT, Ploegh HL (eds) Current protocols in protein science. Wiley, Hoboken, NJ, pp 3.4.1–3.4.29
Okutucu B, Dınçer A, Habib O, Zihnioglu F (2007) Comparison of five methods for determination of total plasma protein concentration. J Biochem Biophys Methods 70(5):709–711
Shen N et al (2015) Highly selective method for rapeseed protein content determination. J Chin Cereals Oils Assoc 2:107–111
Davies C (2005) Introduction to immunoassay principles. In: Wild D (ed) The immunoassay handbook, 3rd edn. Elsevier, London, pp 3–40
Walsh G (2002) Protein purification and characterization. In: Proteins: biochemistry and biotechnology. Wiley, Hoboken, NJ, pp 89–178
Anicetti VR, Keytand BA, Hancock WS (1989) Purity analysis of protein pharmaceuticals produced by recombinant DNA technology. Trends Biotechnol 7(12):342–349
Ernst O, Zor T (2010) Linearization of the Bradford protein assay. J Vis Exp 38:1918. doi:10.3791/1918
López JM, Imperial S, Valderrama R, Navarro S (1993) An improved Bradford protein assay for collagen proteins. Clin Chim Acta 220(1):91–100
Kruger NJ (2009) The Bradford method for protein quantitation. In: Walker JM (ed) The protein protocols handbook, 3rd edn. Humana Press, New York, pp 17–24
Lin AV (2015) Indirect ELISA. MethodsMol Biol 1318:51–59. doi:10.1007/978-1-4939-2742-5_5
Schwab M (2012) Sandwich ELISA. In: Schwab M (ed) Encyclopedia of cancer. Springer, Berlin, pp 3334–3334. doi:10.1007/978-3-642-16483-5_5155
Lin AV (2015) Direct ELISA. Methods Mol Biol 1318:61–67. doi:10.1007/978-1-4939-2742-5_5
Meyer T (2010) Competitive ELISA. In: Kontermann R, Dübel S (eds) Antibody engineering, vol 1. Springer, Berlin, pp 739–742
Rabilloud T (2010) Variations on a theme: changes to electrophoretic separations that can make a difference. J Proteomics 73(8):1562–1572. doi:10.1016/j.jprot.2010.04.001
Dyballa N, Metzger S (2012) Fast and sensitive coomassie staining in quantitative proteomics. Methods Mol Biol 893:47–59. doi:10.1007/978-1-61779-885-6_4
Zellner M, Babeluk R, Diestinger M, Pirchegger P, Skeledzic S, Oehler R (2008) Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples. Electrophoresis 17:3621–3627. doi:10.1002/elps.200700935
Sano T, Smith CL, Cantor CR (1992) Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258:120–122
Niemeyer CM, Adler M, Wacker R (2007) Detecting antigens by quantitative immune-PCR. Nat Protoc 2:1918–1930
Lind K, Kubista M (2005) Development and evaluation of three real-time immuno-PCR assemblages for quantification of PSA. J Immunol Methods 304:107–116
Wang X, He Q, Xu Y, Liu X, Shu M, Tu Z, Li Y, Wang W, Cao D (2016) Anti-idiotypic VHH phage display-mediated immuno-PCR for ultrasensitive determination of mycotoxin zearalenone in cereals. Talanta 147:410–415. doi:10.1016/j.talanta.2015.09.072
Hansen MC, Nederby L, Henriksen MO-B, Hansen M, Nyvold CG (2014) Sensitive ligand-based protein quantification using immuno-PCR: a critical review of single-probe and proximity ligation assays. Biotechniques 56(5):217–228
Meng XY, Li YS, Zhou Y, Zhang YY, Qiao B, Sun Y, Yang L, Hu P, Lu SY, Ren HL, Zhang JH, Wang XR, Liu ZS (2015) Real-time immuno-PCR for ultrasensitive detection of pyrene and other homologous PAHs. Biosens Bioelectron 70:42–47. doi:10.1016/j.bios.2015.03.019
Sun R, Zhuang H (2015) An ultrasensitive gold nanoparticles improved real-time immuno-PCR assay for detecting diethyl phthalate in foodstuff samples. Anal Biochem 480:49–57. doi:10.1016/j.ab.2015.04.010
Meng XY, Li YS, Zhou Y, Sun Y, Qiao B, Si CC, Hu P, Lu SY, Ren HL, Liu ZS, Qiu HJ, Liu JQ (2016) An improved RT-IPCR for detection of pyrene and related polycyclic aromatic hydrocarbons. Biosens Bioelectron 78:194–199. doi:10.1016/j.bios.2015.11.037
Ku H-K, Lim HM, Oh KH, Yang HJ, Jeong JS, Kim SK (2013) Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models. Anal Biochem 434(1):178–180. doi:10.1016/j.ab.2012.10.045
Akins RE, Tuan RS (2009) Ultrafast protein determinations using microwave enhancement. In: Walker JM (ed) The protein protocols handbook, 3rd edn. Humana Press, New York, pp 25–34
Walker JM (2009) SDS polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) The protein protocols handbook, 3rd edn. Humana Press, New York, pp 177–185
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Campion, E.M., Loughran, S.T., Walls, D. (2017). Protein Quantitation and Analysis of Purity. In: Walls, D., Loughran, S. (eds) Protein Chromatography. Methods in Molecular Biology, vol 1485. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6412-3_12
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DOI: https://doi.org/10.1007/978-1-4939-6412-3_12
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