Abstract
Several techniques have been employed to detect proteins on membranes. These include the use of quantum dot luminescent labels, oxyblot immunochemical detection, polymer immunocomplexes, “coupled” probing approach, in situ renaturation of proteins for detecting enzyme activities in crude or purified preparations, immunochromatographic assay, western-phosphatase assay, and use of Congo red dye (a cosmetic color named Alta), Pro-Q Emerald 488 dye, or amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates for the simultaneous trichromatic fluorescence detection of proteins. Several methods have been used to improve the detection of proteins on membranes, including glutaraldehyde treatment of nitrocellulose blots, elimination of keratin artifacts in immunoblots probed with polyclonal antibodies, and washing of immunoblots with excessive water and manipulation of Tween-20 in wash buffer. These methods are briefly reviewed in this chapter.
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References
Kurien BT, Hensley K, Bachmann M, Scofield RH (2006) Oxidatively modified autoantigens in autoimmune diseases. Free Radic Biol Med 41:549–556
Castegna A, Aksenov M, Thongboonkerd V, Klein JB, Pierce WM, Booze R et al (2002) Proteomic identification of oxidatively modified proteins in Alzheimer’s disease brain. Part II: dihydropyrimidinase-related protein 2, alpha-enolase and heat shock cognate 71. J Neurochem 82:1524–1532
Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 324:1–18, Review
Davies KJ (2001) Degradation of oxidized proteins by the 20S proteasome. Biochimie 83:301–310, Review
Stadtman ER (1990) Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic Biol Med 9:315–325, Review. Erratum in Free Radic Biol Med. 10: 249
Aksenov M, Aksenova M, Butterfield DA, Markesbery WR (2000) Oxidative modification of creatine kinase BB in Alzheimer’s disease brain. J Neurochem 74:2520–2527
Krajewski S, Zapata JM, Reed JC (1996) Detection of multiple antigens on western blots. Anal Biochem 236:221–228
Gingrich JC, Davis DR, Nguyen Q (2000) Multiplex detection and quantitation of proteins on western blots using fluorescent probes. Biotechniques 29:636–642
Makrides SC, Gasbarro C, Bello JM (2005) Bioconjugation of quantum dot luminescent probes for Western blot analysis. Biotechniques 39:501–506
Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP et al (2003) Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol 21:41–46
Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A (2002) In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298:1759–1762
Martin K, Hart C, Liu J, Leung WY, Patton WF (2003) Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates. Proteomics 3:1215–1227
Fukuda T, Tani Y, Kobayashi T, Hirayama Y, Hino O (2000) A new Western blotting method using polymer immunocomplexes: detection of Tsc1 and Tsc2 expression in various cultured cell lines. Anal Biochem 285:274–276
Wu M, Stockley PG, Martin WJ 2nd (2002) An improved Western blotting technique effectively reduces background. Electrophoresis 23:2373–2376
Morrisey JH (1981) Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310
Dunbar BS (1987) Two dimensional electrophoresis immunological techniques. Plenum Press, New York, NY, p 74
Ocha D (1983) Protein contaminants of sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem 135:470–474
Bérubé B, Coutu L, Lefièvre L, Bégin S, Dupont H et al (1994) The elimination of keratin artifacts in immunoblots probed with polyclonal antibodies. Anal Biochem 217:331–333
Sundaram P (1996) A simplified ‘coupled’ probing approach for the detection of proteins on dot and western blots. J Immunol Methods 195:149–152
Béguin P (1983) Detection of cellulase activity in polyacrylamide gels using Congo red-stained agar replicas. Anal Biochem 131:333–336
Mehta S, Rajput YS (1998) A method for staining of proteins in nitrocellulose membrane and acrylamide gel using Congo red dye. Anal Biochem 263:248–251
Pal JK, Godbole D, Sharma K (2004) Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic. J Biochem Biophys Methods 61:339–347
Shackelford DA, Zivin JA (1993) Renaturation of calcium/calmodulin-dependent protein kinase activity after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to membranes. Anal Biochem 211:131–138
Hart C, Schulenberg B, Steinberg TH, Leung WY, Patton WF (2003) Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24:588–598
Steinberg TH, Pretty On Top K, Berggren KN, Kemper C, Jones L, Diwu Z et al (2001) Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots. Proteomics 1:841–855
Paek SH, Lee SH, Cho JH, Kim YS (2000) Development of rapid one-step immunochromatographic assay. Methods 22:53–60
Maya R, Oren M (2000) Unmasking of phosphorylation-sensitive epitopes on p53 and Mdm2 by a simple Western-phosphatase procedure. Oncogene 19:3213–3215
Dominguez DC, Adams H, Hageman JH (1999) Immunocytochemical localization of a calmodulin like protein in Bacillus subtilis cells. J Bacteriol 181:4605–4610
Liang FT, Granstrom DE, Shi YF (1997) Concentrating protein samples for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing using protein-blotting membranes. J Chromatogr A 764:143–150
Spinola SM, Cannon JG (1985) Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes. J Immunol Methods 81:161–165
Batteiger B, Newhall WJ 5th, Jones RB (1982) The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods 55:297–307
Lin W, Kasamatsu H (1983) On the electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal Biochem 128:302–311
Hoffman WL, Jump AA, Ruggles AO (1994) Soaking nitrocellulose blots in acidic buffers improves the detection of bound antibodies without loss of biological activity. Anal Biochem 217:153–155
Kurien BT, Scofield RH (2009) A brief review of other notable protein detection methods. Methods Mol Biol 536:557–571, Review
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Kurien, B.T., Scofield, R.H. (2015). Other Notable Methods of Membrane Protein Detection: A Brief Review. In: Kurien, B., Scofield, R. (eds) Detection of Blotted Proteins. Methods in Molecular Biology, vol 1314. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2718-0_36
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DOI: https://doi.org/10.1007/978-1-4939-2718-0_36
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