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Fluorescent enzyme assay methods and compositions

A technology of composition and fluorescent part, which is applied in the field of luciferase analysis and composition, and can solve the problems of inconvenient and expensive analysis operations

Inactive Publication Date: 2005-11-16
阿普尔拉股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available assays are inconvenient, expensive, or have other drawbacks

Method used

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  • Fluorescent enzyme assay methods and compositions
  • Fluorescent enzyme assay methods and compositions
  • Fluorescent enzyme assay methods and compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0245] Protein Kinase Assay

[0246] a. Protein Kinase Substrate (Compound 1)

[0247] An exemplary enzyme substrate for the detection of protein kinase A, palmitoyl-FAM-S (OPO 3 2- )LRRRRFSK(Ac)G-amide. Use standard FastMoc TM Chemical method Construction of peptide Fmoc-L(R(Pmc)) via solid-phase peptide synthesis on 625mg of Fmoc-PAL-PEG-PS resin at 0.16mmol / g 4 FS(tBu)K(Mtt)G, the resin is a solid support for generating carboxyamide peptides. A portion of the final protected peptide-resin (20 mg, 2 μmol peptide) was transferred to a 1.5 ml Eppendorf tube and treated with 1 mL of 5% trifluoroacetic acid (TFA) in dichloromethane (DCM) to generate the characteristic yellow trityl base color. The resin was precipitated by adding 0.1 mL methanol and washing (3 x 1 mL dimethylformamide, DMF). Diisopropylethylamine (50 μL) and capping solution (1 mL of acetic anhydride solution (0.5 M) and hydroxybenzotriazole (0.015 M) in N-methylpyrrolidone (NMP)) were added to the re...

Embodiment 2

[0251] Phosphatase detection

[0252] a. Phosphatase substrate (compound 2)

[0253] An exemplary dye-labeled peptide is described below, compound 2, palmitoyl-LRRRFS (OPO 3 2- ) Synthesis of K(5-FAM)G-amide. Use standard FastMoc TM Chemical method Construction of peptide Fmoc-LR(Pmc) via solid-phase peptide synthesis at 0.16mmol / g on 625mg of Fmoc-PAL-PEG-PS resin 4 FS(OPO(OBzl)OH)K(Mtt)G, the resin is a solid support for generating carboxyamide peptides. Peptides with carboxyl termini were constructed with Fmoc-Gly-PEG-PS resin. A portion of the final protected peptide-resin (20 mg, 2 μmol peptide) was transferred to a 1.5 ml Eppendorf tube and treated with 1 mL of 5% trifluoroacetic acid (TFA) in dichloromethane (DCM) to generate the characteristic yellow triphenyl Methyl color. The resin was precipitated by adding 0.1 mL methanol and washing (3 x 1 mL dimethylformamide, DMF). 5-Carboxyfluorescein succinimidyl ester (5 mg, 10 μmol), diisopropylethylamine (30 μL,...

Embodiment 3

[0257] Fluorescence dynamic range

[0258] This example describes a study to determine the difference in fluorescence between phosphorylated and non-phosphorylated forms of a protein kinase A substrate. The goal is to determine the extent to which a compound provides an increase in fluorescent signal with a good signal-to-noise ratio after phosphorylation of the non-phosphorylated form.

[0259] a. Candidate substrates (compounds 3, 3P, 4, 4P, 5, 5P, 6 and 6P)

[0260] A series of compounds with alkylacyl groups of varying lengths (Scheme 3 above) were prepared in both phosphorylated and non-phosphorylated form, represented by the formula: X-Y(dye)LRRAS(OR)LG-NH 2 , where X is CH 3 (CH 2 ) x C(=O)-form fatty acid acyl group, x is 0, 7, 10 or 14, Y is α-aminomethylglycine, the dye is attached to 4,7-dichlorofluorescein at the 2-amino nitrogen atom of Y, and R is H or PO 3 2- .

[0261] For compounds 3-6, standard FastMoc TM Chemical method Construction of peptide F...

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Abstract

Disclosed are fluorescent compositions and methods for detecting and / or characterizing enzymes and various uses thereof.

Description

[0001] related application [0002] This application claims priority under 35 U.S.C. §119(e) to the following applications: Application Serial No. 60 / 409,178, titled "Luciferase Assay Methods and Compositions," filed September 9, 2002; and Application Serial Serial No. 60 / 486,393, entitled "Luciferase Assay Methods and Compositions," filed July 10, 2003, the disclosure of which application is incorporated herein by reference in its entirety. field of invention [0003] The present invention relates to fluorescent compositions and methods for detecting or characterizing enzymes and various uses thereof. Background technique [0004] Enzyme analysis is an important tool for studying and testing enzymes for biological and industrial applications. In living organisms, enzymes perform various tasks such as synthesis and replication of nucleic acids, modification and degradation of polypeptides, synthesis of metabolites, and many other functions. Enzymes are also used in industr...

Claims

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Application Information

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IPC IPC(8): C12Q1/00
Inventor 罗纳德·J·格雷厄姆林达·G·李理查德·L·诺布尔孙洪业
Owner 阿普尔拉股份有限公司
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