Fluorescent ellman assay for free thiol detection
AssigneeGenentech, Inc.
InventorsAron Lee, Rashmi Sharma, Justin Joo-Ho Jeong, Michael Tae-Jon Kim
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Abstract
The present disclosure relates to methods and kits for detecting a free thiol in a substrate as well as methods for quantifying the amount of free thiol in a substrate. In particular, the present disclosure provides a fluorescent Ellman assay for enhanced sensitivity of free thiol detection and quantification.
Claims34
A method for detecting a free thiol, the method comprising:
- a) contacting a thiol substrate with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichimetrically liberate TNB2−;
- b) contacting the liberated TNB2− with a reagent that interacts with the liberated TNB2− to produce a fluorescent signal;
- and
- c) detecting the fluorescent signal emitted by the interaction of the liberated TNB2− with the reagent to thereby detect the free thiol.
The method of 1, wherein the reagent is a fluorogenic probe or a fluorescent probe, wherein incubating the TNB2− molecules with the fluorogenic probe or the fluorescent probe results in formation of:
- a) a fluorescent TNB-probe adduct;
- or
- b) a deprotected fluorescent probe and a non-fluorescent TNB adduct.
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A method of quantifying the free thiol content of a thiol substrate, the method comprising:
- a) contacting the thiol substrate with 5.5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to stoichiometrically liberate TNB2−;
- b) incubating the TNB2− with a reagent that interacts with the liberated TNB2− to produce a fluorescent signal;
- c) detecting a fluorescent signal emitted by the interaction of the liberated TNB2− with the reagent;
- and
- d) quantifying the free thiol content of the molecule by comparing the signal detected in c) with a reference signal of known quantity.
The method of 19, wherein the reagent is a fluorogenic probe or a fluorescent probe, wherein incubation of TNB2− with the fluorogenic or fluorescent probe results in formation of:
- a) a fluorescent TNB-probe adduct;
- or
- b) a deprotected fluorescent probe and a non-fluorescent TNB adduct.
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A kit for detecting a thiol compound, comprising:
- a) 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB);
- and
- b) a fluorogenic probe or a fluorescent probe.
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Dependencies are inferred from the claim text.