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An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels.
Electrophoresis. 2001 Aug; 22(13):2646-52.E

Abstract

A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology.

Authors+Show Affiliations

Visal-Shah S
Département de Phytologie, Centre de Recherche en Horticulture, Université Laval, Québec, Canada.
Vrain TC
No affiliation info available
Yelle TC
No affiliation info available
Nguyen-Quoc B
No affiliation info available
Michaud D
No affiliation info available

MeSH

Acrylic ResinsAnimalsBromelainsCattleElectrophoresis, Polyacrylamide GelEndopeptidasesGelatinProtease Inhibitors

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11545387

Citation

Visal-Shah, S, et al. "An Electroblotting, Two-step Procedure for the Detection of Proteinases and the Study of Proteinase/inhibitor Complexes in Gelatin-containing Polyacrylamide Gels." Electrophoresis, vol. 22, no. 13, 2001, pp. 2646-52.
Visal-Shah S, Vrain TC, Yelle TC, et al. An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels. Electrophoresis. 2001;22(13):2646-52.
Visal-Shah, S., Vrain, T. C., Yelle, T. C., Nguyen-Quoc, B., & Michaud, D. (2001). An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels. Electrophoresis, 22(13), 2646-52.
Visal-Shah S, et al. An Electroblotting, Two-step Procedure for the Detection of Proteinases and the Study of Proteinase/inhibitor Complexes in Gelatin-containing Polyacrylamide Gels. Electrophoresis. 2001;22(13):2646-52. PubMed PMID: 11545387.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - An electroblotting, two-step procedure for the detection of proteinases and the study of proteinase/inhibitor complexes in gelatin-containing polyacrylamide gels. AU - Visal-Shah,S, AU - Vrain,T C, AU - Yelle,T C, AU - Nguyen-Quoc,B, AU - Michaud,D, PY - 2001/9/8/pubmed PY - 2002/1/29/medline PY - 2001/9/8/entrez SP - 2646 EP - 52 JF - Electrophoresis JO - Electrophoresis VL - 22 IS - 13 N2 - A two-step gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE) procedure was devised for the detection of proteinases and the study of proteinase/inhibitor interactions in complex biological extracts. The proteins are first resolved by sodium dodecyl sulfate (SDS)-PAGE under reducing or nonreducing conditions, and electrotransferred into a 0.75 mm-thick accompanying polyacrylamide slab gel containing 0.1% w/v porcine gelatin. The active proteinase bands are developed by a gelatin proteolysis step in the accompanying gel in the presence or absence of diagnostic proteinase inhibitors, allowing the assessment of proteinase classes and the visual discrimination of inhibitor-'sensitive' and -'insensitive' proteinases in complex extracts. Alternatively, protein extracts are preincubated with specific reversible inhibitors before electrophoresis, allowing a rapid discrimination of strong and weak interactions implicating proteinases and reversible inhibitors. In comparison with the standard gelatin/PAGE procedure, that involves copolymerization of gelatin with acrylamide in the resolving gel, this new procedure simplifies proteinase patterns, avoids overestimation of proteinase numbers in complex extracts, and allows in certain conditions the estimation of proteinase molecular weights. Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), papain (EC 3.4.22.2), and the extracellular (digestive) cysteine proteinases of five herbivorous pests are used as model enzymes to illustrate the usefulness of this approach in detecting proteinases and in studying their interactions with specific proteinaceous inhibitors potentially useful in biotechnology. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/11545387/An_electroblotting_two_step_procedure_for_the_detection_of_proteinases_and_the_study_of_proteinase/inhibitor_complexes_in_gelatin_containing_polyacrylamide_gels_ L2 - https://doi.org/10.1002/1522-2683(200108)22:13<2646::AID-ELPS2646>3.0.CO;2-8 DB - PRIME DP - Unbound Medicine ER -