Tags

Type your tag names separated by a space and hit enter

Active protease mapping in 2DE gels.
Methods Mol Biol. 2009; 519:431-8.MM

Abstract

Proteases act as the molecular mediators of many vital biological processes. To understand the function of each protease, it needs to be separated from other proteins and characterized in its natural, biologically active form. In the method described in this chapter, proteases in a biological sample are separated under nonreducing conditions in 2DE gels. A specific small protease substrate, tagged with a fluorescent dye, is copolymerized into the SDS gel in the second dimension. After electrophoresis, the proteins are renatured by washing the gel with Triton X-100 solution or Milli Q water to remove SDS. The gel is then incubated in a protease assay buffer. The hydrolysis of the tagged specific substrate by the renatured protease releases the free fluorescent dye, which fluoresces in situ. The fluorescent spots indicate the location of the specific proteases in the gel and the specificity of the proteases.

Links

Publisher Full Text (DOI)

Authors+Show Affiliations

Zhao Z
Institute for Eye Research and Vision Cooperative Research Centre, University of New South Wales, Sydney, NSW, Australia.
Russell PJ
No affiliation info available

MeSH

Electrophoresis, Gel, Two-DimensionalFluorescent DyesHydrogen-Ion ConcentrationIsoelectric FocusingPeptide Hydrolases

Pub Type(s)

Journal Article

Language

eng

PubMed ID

19381600

Citation

Zhao, Zhenjun, and Pamela J. Russell. "Active Protease Mapping in 2DE Gels." Methods in Molecular Biology (Clifton, N.J.), vol. 519, 2009, pp. 431-8.
Zhao Z, Russell PJ. Active protease mapping in 2DE gels. Methods Mol Biol. 2009;519:431-8.
Zhao, Z., & Russell, P. J. (2009). Active protease mapping in 2DE gels. Methods in Molecular Biology (Clifton, N.J.), 519, 431-8. https://doi.org/10.1007/978-1-59745-281-6_28
Zhao Z, Russell PJ. Active Protease Mapping in 2DE Gels. Methods Mol Biol. 2009;519:431-8. PubMed PMID: 19381600.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Active protease mapping in 2DE gels. AU - Zhao,Zhenjun, AU - Russell,Pamela J, PY - 2009/4/22/entrez PY - 2009/4/22/pubmed PY - 2009/6/3/medline SP - 431 EP - 8 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol Biol VL - 519 N2 - Proteases act as the molecular mediators of many vital biological processes. To understand the function of each protease, it needs to be separated from other proteins and characterized in its natural, biologically active form. In the method described in this chapter, proteases in a biological sample are separated under nonreducing conditions in 2DE gels. A specific small protease substrate, tagged with a fluorescent dye, is copolymerized into the SDS gel in the second dimension. After electrophoresis, the proteins are renatured by washing the gel with Triton X-100 solution or Milli Q water to remove SDS. The gel is then incubated in a protease assay buffer. The hydrolysis of the tagged specific substrate by the renatured protease releases the free fluorescent dye, which fluoresces in situ. The fluorescent spots indicate the location of the specific proteases in the gel and the specificity of the proteases. SN - 1064-3745 UR - https://www.unboundmedicine.com/medline/citation/19381600/Active_protease_mapping_in_2DE_gels_ L2 - https://dx.doi.org/10.1007/978-1-59745-281-6_28 DB - PRIME DP - Unbound Medicine ER -