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Two-Dimensional Zymography of Proteases from Steatotic Duck Liver

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Zymography

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1626))

Abstract

Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

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Abbreviations

2DE:

Two dimensional electrophoresis

2DZ:

Two dimensional zymography

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

IEF:

Isoelectric focusing

MMP:

Matrix metalloproteases

PAG:

Polyacrylamide gel

PAGE:

Polyacrylamide gel electrophoresis

pI :

Isoelectric point

PMSF:

Phenylmethyl sulfonyl fluoride

SDS:

Sodium dodecyl sulphate

TCA:

Trichloroacetic acid

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Acknowledgments

We thank INP-ENSAT for supporting the stay of Dr. Wilkesman at GenPhySE, Université de Toulouse, as well as the Consejo de Desarrollo Científico y Humanísitico de la Universidad de Carabobo, Venezuela, for partial funding of this research (CDCH-AM-030-11, CDCH-187-2015). The authors thank the staff of GenPhySE lab for technical assistance and Dr. Molette for her useful comments.

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Authors and Affiliations

  1. Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología, Universidad de Carabobo, Valencia, 2005, Venezuela

    Jeff Wilkesman & Liliana Kurz

  2. GenPhySE, Université de Toulouse, INRA, INPT, ENVT, Castanet Tolosan, France

    Hervé Rémignon

  3. Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología, Universidad de Carabobo, Valencia, Venezuela

    María Fernanda Padrón

Authors
  1. Jeff Wilkesman
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  2. María Fernanda Padrón
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  3. Liliana Kurz
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  4. Hervé Rémignon
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Corresponding author

Correspondence to Jeff Wilkesman .

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Editors and Affiliations

  1. Centre for Environmental, Biology and Chemistry Research, Facultad de Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela

    Jeff Wilkesman

  2. Centre for Environmental, Biology and Chemistry Research Facultad de Ciencias y Tecnología, University of Carabobo, Valencia, Venezuela

    Liliana Kurz

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Wilkesman, J., Padrón, M.F., Kurz, L., Rémignon, H. (2017). Two-Dimensional Zymography of Proteases from Steatotic Duck Liver. 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_14

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  • DOI: https://doi.org/10.1007/978-1-4939-7111-4_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7109-1

  • Online ISBN: 978-1-4939-7111-4

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