Abstract
Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.
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References
Gupta R, Beg QK, Lorenz P (2002) Bacterial alkaline proteases: molecular approaches and industrial applications. Appl Microbiol Biotechnol 59:15–32
Godfrey T, West S (1996) Introduction to industrial enzymology. In: Godfrey T, West S (eds) Industrial enzymology, 2nd edn. Macmillan, London, pp 1–8
Kim SH, Choi NS, Lee WY (1998) Fibrin zymography: a direct analysis of fibrinolytic enzymes on gels. Anal Biochem 263:115–116
Kim SH, Choi NS (1999) Electrophoretic analysis of protease inhibitors in fibrin zymography. Anal Biochem 270:179–181
Choi NS, Kim SH (2000) Two fibrin zymography methods for analysis of plasminogen activators on gels. Anal Biochem 281:236–238
Chung DM, Kim KE, Ahn KH et al (2011) Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel. Biotechnol Lett 33:1663–1666
Brown TL, Yet MG, Wold F (1982) Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes. Anal Biochem 122:164–172
Lantz MS, Ciborowski P (1994) Zymographic techniques for detection and characterization of microbial proteases. Methods Enzymol 235:563–594
Laemmli UK (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685
Choi NS, Kim BH, Park CS et al (2009) Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel. Anal Biochem 386:121–122
Choi NS, Choi JH, Han YJ et al (2009) Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel. Electrophoresis 30:2234–2237
Hames BD (1981) Gel electrophoresis of proteins. In: Hames BD, Rickwood D (eds) A practical approach. IRL Press, London, p 290
Park CS, Kang DO, Lee WY et al (2015) Identification of two types binding modes using reverse or diagonal electrophoretic zymography. Acad J Biotechnol 3:52–55
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant number 2013R1A1A2062475).
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Park, CS., Kang, DO., Choi, NS. (2017). Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel. In: Wilkesman, J., Kurz, L. (eds) Zymography. Methods in Molecular Biology, vol 1626. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7111-4_16
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DOI: https://doi.org/10.1007/978-1-4939-7111-4_16
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Publisher Name: Humana Press, New York, NY
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