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Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples.
Electrophoresis. 2003 Sep; 24(18):3284-8.E

Abstract

To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies.

Authors+Show Affiliations

Oncology Research Centre, Prince of Wales Hospital of Sydney, Randwick, NSW, Australia.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

14518058

Citation

Zhao, Zhenjun, and Pamela J. Russell. "Trypsin Activity Assay in Substrate-specific One- and Two-dimensional Gels: a Powerful Method to Separate and Characterize Novel Proteases in Active Form in Biological Samples." Electrophoresis, vol. 24, no. 18, 2003, pp. 3284-8.
Zhao Z, Russell PJ. Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples. Electrophoresis. 2003;24(18):3284-8.
Zhao, Z., & Russell, P. J. (2003). Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples. Electrophoresis, 24(18), 3284-8.
Zhao Z, Russell PJ. Trypsin Activity Assay in Substrate-specific One- and Two-dimensional Gels: a Powerful Method to Separate and Characterize Novel Proteases in Active Form in Biological Samples. Electrophoresis. 2003;24(18):3284-8. PubMed PMID: 14518058.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples. AU - Zhao,Zhenjun, AU - Russell,Pamela J, PY - 2003/10/1/pubmed PY - 2004/6/15/medline PY - 2003/10/1/entrez SP - 3284 EP - 8 JF - Electrophoresis JO - Electrophoresis VL - 24 IS - 18 N2 - To separate and identify the proteases, a substrate-specific, sensitive assay in sodium dodecyl sulfate (SDS)-polyacrylamide gels after two-dimensional (2-D) electrophoresis has been developed. This method allows simultaneous determination of protease cleavage specificity, molecular weight, isoelectric point, and if necessary, amino acid sequencing. After isoelectric focusing in immobilized pH gradient (IPG) strips (pH 6-11) (first dimension), trypsin was electrophoresed in 12% SDS polyacrylamide gels (second dimension) copolymerized with Boc-Gln-Ala-Arg-MCA (4-methyl-coumaryl-7-amide). The gels were washed in cold 2.5% Triton X-100 and water, and incubated in assay buffer (6.3 mM Bicine, 100 mM NaCl). Trypsin cleavage of the peptide-MCA generated fluorescent 7-amino-4-methyl-coumarin. In 1-D gels, as low as 500 pg trypsin could be detected and trypsin band volumes correlated linearly with the amounts of trypsin (R(2) = 0.999). In 2-D gels, the lowest amount of trypsin detected was 1 ng. The linear regression of spot volume and loading amount was still good (R(2) = 0.974). To optimize renaturation conditions, 5x5 min washes with 2.5% Triton X-100 and water, respectively, gave the strongest band volume. For fluorescence development, an assay buffer at pH 9 was the best; incubation at 37 degrees C for 30 min was sufficient. The method has application for identifying novel proteases as it does not rely on antibodies. SN - 0173-0835 UR - https://www.unboundmedicine.com/medline/citation/14518058/Trypsin_activity_assay_in_substrate_specific_one__and_two_dimensional_gels:_a_powerful_method_to_separate_and_characterize_novel_proteases_in_active_form_in_biological_samples_ DB - PRIME DP - Unbound Medicine ER -