CN110412004A - A kind of activity test method of deubiquitinating enzymes - Google Patents
A kind of activity test method of deubiquitinating enzymes Download PDFInfo
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- CN110412004A CN110412004A CN201910806260.0A CN201910806260A CN110412004A CN 110412004 A CN110412004 A CN 110412004A CN 201910806260 A CN201910806260 A CN 201910806260A CN 110412004 A CN110412004 A CN 110412004A
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- deubiquitinating enzymes
- substrate
- reaction buffer
- working solution
- dtt
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Abstract
The invention discloses a kind of activity test methods of deubiquitinating enzymes, comprising the following steps: S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room temperature, is protected from light；S2, the 1X reaction buffer containing 10mM DTT is prepared, and deubiquitinating enzymes working solution and substrate working solution is prepared respectively with the 1X reaction buffer containing 10mM DTT；Compared with conventional fluorescent analytic approach, the chemiluminescence method in the present invention shows the broader range of linearity, higher sensitivity and lower detection limit, therefore can obtain the detection data of higher reliability；Under the premise of obtaining comparable results, by Aminofluorescein label ubiquitin (Ub-AML) as enzyme amount required for luminous substrate, several hundred lower than enzyme amount needed for conventional fluorescent method or even thousands of times, to greatly reduce the cost of high-flux medicaments sifting.
Description
Technical field
The invention belongs to deubiquitinating enzymes technical fields, and in particular to a kind of activity test method of deubiquitinating enzymes.
Background technique
Deubiquitinating enzymes are a kind of large numbers of protease, in many screening experiment rooms, at present using twenty years ago
The activity for the measuring method measurement deubiquitinating enzymes based on steady-state fluorescence established.The fluorogenic substrate that this method uses, such as 7-
Amino -4- methylcoumarin marks ubiquitin protein (Ub-AMC) or rhodamine 110 to mark ubiquitin protein (Ub-Rho).These bottoms
Object is effectively cracked or is hydrolyzed by various deubiquitinating enzymes, releases the part of high fluorescent.The measurement has been used for various removing ubiquitin
Change inhibitor screening, such as identifying USP1 and USP7 inhibitor.
Have the shortcomings that one it is significant, especially for Ub-AMC, be they be susceptible to many small molecules show it is glimmering
Light is interfered；In addition, due to the ubiquitin conjugate formed under the connection of ubiquitin and fluorophor in these substrates and physiological condition
In isopeptide bond architectural difference it is larger so that many deubiquitinating enzymes are very slow to their identification and processing speed, thus
Greatly reduce the measurement method sensitivity and experimental data reliability the problem of, thus it is proposed that a kind of deubiquitination
The activity test method of enzyme.
Summary of the invention
The purpose of the present invention is to provide a kind of activity test methods of deubiquitinating enzymes, to solve in above-mentioned background technique
The problem of proposition.
To achieve the above object, the invention provides the following technical scheme: a kind of activity test method of deubiquitinating enzymes, packet
Include following steps:
S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room
Temperature is protected from light；
S2, the 1X reaction buffer containing 10mM DTT is prepared, and with the 1X reaction buffer difference containing 10mM DTT
Prepare deubiquitinating enzymes working solution and substrate working solution；
S3, in white 96 orifice plates, following volumes component is added to obtain the mixed liquor of the total reaction volume of 20ul: a-
10ul 1X reaction buffer；B-5ul deubiquitinating enzymes working solution；C-5ul substrate working solution；
S4, slightly be centrifuged white 96 orifice plates makes each component solution gather bottom hole mixes well, by S3 gained mixed liquor
Reaction is placed in incubation at room temperature 25-35 minutes；
S5,20ul fluorescein detection reagent is added in the every hole of white 96 orifice plates, 96 orifice plates of white is then placed in shaking table
On rock 2 minutes, 96 orifice plates of white are placed in room temperature later and continue to be incubated for 30 minutes；
S6, disk-read record the luminous value in every hole.
Preferably, the substrate is that Aminofluorescein marks ubiquitin (Ub-AML), and the concentration of substrate is 0.6uM.
Preferably, the 1X reaction buffer in the S2 containing 10mM DTT is the reaction buffer after thawing in S1
On the basis of be diluted to obtain.
Preferably, the deubiquitinating enzymes include cysteine and all six other deubiquitinating enzymes of metalloproteinases
Family.
Preferably, pass through will be containing 10mM DTT's for deubiquitinating enzymes working solution and substrate working solution in the S2
1X reaction buffer is poured into the glassware for filling deubiquitination enzyme-to-substrate respectively and is made.
Compared with prior art, the beneficial effects of the present invention are: in the present invention, by the way that Aminofluorescein is marked ubiquitin
(Ub-AML), as the substrate of deubiquitinating enzymes, the catalytic action of deubiquitination releases one kind from substrate, and to be called amino glimmering
The small molecule of light element (AML), it is in ATP and MgCl2In the presence of with recombination luciferase reaction generate it is bright shine, with tradition
Fluorescence analysis is compared, and the chemiluminescence method in the present invention shows the broader range of linearity, higher sensitivity and lower
Detection limit, therefore the detection data of higher reliability can be obtained；Under the premise of obtaining comparable results, pass through Aminofluorescein
Mark ubiquitin (Ub-AML) as enzyme amount required for luminous substrate, it is several hundred or even thousands of lower than enzyme amount needed for conventional fluorescent method
Times, to greatly reduce the cost of high-flux medicaments sifting.
Detailed description of the invention
Fig. 1 is the Activity determination result curve figure of deubiquitinating enzymes of the invention.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Embodiment 1
The present invention provides a kind of technical solution: a kind of activity test method of deubiquitinating enzymes, comprising the following steps:
S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room
Temperature is protected from light；
S2, the 1X reaction buffer containing 10mM DTT is prepared, and with the 1X reaction buffer difference containing 10mM DTT
Prepare deubiquitinating enzymes working solution and substrate working solution；
S3, in white 96 orifice plates, following volumes component is added to obtain the mixed liquor of the total reaction volume of 20ul: a-
10ul 1X reaction buffer；B-5ul deubiquitinating enzymes working solution；C-5ul substrate working solution, if doing high flux screening,
It needs for deubiquitinating enzymes and compound to be screened to be incubated in advance 15 minutes；
S4, slightly be centrifuged white 96 orifice plates makes each component solution gather bottom hole mixes well, by S3 gained mixed liquor
Reaction is placed in incubation at room temperature 25 minutes；
S5,20ul fluorescein detection reagent is added in the every hole of white 96 orifice plates, 96 orifice plates of white is then placed in shaking table
On rock 2 minutes, 96 orifice plates of white are placed in room temperature later and continue to be incubated for 30 minutes；
S6, disk-read record the luminous value in every hole.
Wherein, substrate is that Aminofluorescein marks ubiquitin (Ub-AML), and concentration of substrate is 0.6uM.
Wherein, on the basis of the 1X reaction buffer in S2 containing 10mM DTT is the reaction buffer after thawing in S1
It is diluted to obtain.
Wherein, deubiquitinating enzymes include cysteine and all six other deubiquitination enzyme families of metalloproteinases.
Wherein, deubiquitinating enzymes working solution passes through with substrate working solution and reacts the 1X containing 10mM DTT in S2
Buffer is poured into the glassware for filling deubiquitination enzyme-to-substrate respectively and is made.
Embodiment 2
The present invention provides a kind of technical solution: a kind of activity test method of deubiquitinating enzymes, comprising the following steps:
S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room
Temperature is protected from light；
S2, the 1X reaction buffer containing 10mM DTT is prepared, and with the 1X reaction buffer difference containing 10mM DTT
Prepare deubiquitinating enzymes working solution and substrate working solution；
S3, in white 96 orifice plates, following volumes component is added to obtain the mixed liquor of the total reaction volume of 20ul: a-
10ul 1X reaction buffer；B-5ul deubiquitinating enzymes working solution；C-5ul substrate working solution, if doing high flux screening,
It needs for deubiquitinating enzymes and compound to be screened to be incubated in advance 15 minutes；
S4, slightly be centrifuged white 96 orifice plates makes each component solution gather bottom hole mixes well, by S3 gained mixed liquor
Reaction is placed in incubation at room temperature 30 minutes；
S5,20ul fluorescein detection reagent is added in the every hole of white 96 orifice plates, 96 orifice plates of white is then placed in shaking table
On rock 2 minutes, 96 orifice plates of white are placed in room temperature later and continue to be incubated for 30 minutes；
S6, disk-read record the luminous value in every hole.
Wherein, substrate is that Aminofluorescein marks ubiquitin (Ub-AML), and concentration of substrate is 0.6uM.
Wherein, on the basis of the 1X reaction buffer in S2 containing 10mM DTT is the reaction buffer after thawing in S1
It is diluted to obtain.
Wherein, deubiquitinating enzymes include cysteine and all six other deubiquitination enzyme families of metalloproteinases.
Wherein, deubiquitinating enzymes working solution passes through with substrate working solution and reacts the 1X containing 10mM DTT in S2
Buffer is poured into the glassware for filling deubiquitination enzyme-to-substrate respectively and is made.
Embodiment 3
The present invention provides a kind of technical solution: a kind of activity test method of deubiquitinating enzymes, comprising the following steps:
S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room
Temperature is protected from light；
S2, the 1X reaction buffer containing 10mM DTT is prepared, and with the 1X reaction buffer difference containing 10mM DTT
Prepare deubiquitinating enzymes working solution and substrate working solution；
S3, in white 96 orifice plates, following volumes component is added to obtain the mixed liquor of the total reaction volume of 20ul: a-
10ul 1X reaction buffer；B-5ul deubiquitinating enzymes working solution；C-5ul substrate working solution, if doing high flux screening,
It needs for deubiquitinating enzymes and compound to be screened to be incubated in advance 15 minutes；
S4, slightly be centrifuged white 96 orifice plates makes each component solution gather bottom hole mixes well, by S3 gained mixed liquor
Reaction is placed in incubation at room temperature 35 minutes；
S5,20ul fluorescein detection reagent is added in the every hole of white 96 orifice plates, 96 orifice plates of white is then placed in shaking table
On rock 2 minutes, 96 orifice plates of white are placed in room temperature later and continue to be incubated for 30 minutes；
S6, disk-read record the luminous value in every hole.
Wherein, substrate is that Aminofluorescein marks ubiquitin (Ub-AML), and concentration of substrate is 0.6uM.
Wherein, on the basis of the 1X reaction buffer in S2 containing 10mM DTT is the reaction buffer after thawing in S1
It is diluted to obtain.
Wherein, deubiquitinating enzymes include cysteine and all six other deubiquitination enzyme families of metalloproteinases.
Wherein, deubiquitinating enzymes working solution passes through with substrate working solution and reacts the 1X containing 10mM DTT in S2
Buffer is poured into the glassware for filling deubiquitination enzyme-to-substrate respectively and is made.
Different enzyme amount are made with activity inspection respectively according to the activity test method of deubiquitinating enzymes described in embodiment 1,2,3
It surveys, obtained luminous intensity mean value is as follows:
Curve graph corresponding with this table is shown in Fig. 1；
It can thus be appreciated that the chemiluminescence method in the present invention shows the broader range of linearity, higher sensitivity and lower
Detection limit, therefore the detection data of higher reliability can be obtained；Under the premise of obtaining comparable results, by Aminofluorescein mark
Remember ubiquitin (Ub-AML), enzyme amount required for the substrate as deubiquitinating enzymes is several hundred very lower than enzyme amount needed for conventional fluorescent method
To thousands of times, to greatly reduce the cost of high-flux medicaments sifting.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (5)
1. a kind of activity test method of deubiquitinating enzymes, which comprises the following steps:
S1, deubiquitinating enzymes, substrate and reaction buffer are placed in and are thawed on ice, fluorescein detection reagent is placed in room temperature, is kept away
Light；
S2, the 1X reaction buffer containing 10mM DTT is prepared, and is prepared respectively with the 1X reaction buffer containing 10mM DTT
Deubiquitinating enzymes working solution and substrate working solution；
S3, in white 96 orifice plates, following volumes component is added to obtain the mixed liquor of the total reaction volume of 20ul: a-10ul
1X reaction buffer；B-5ul deubiquitinating enzymes working solution；C-5ul substrate working solution；
S4, slightly be centrifuged white 96 orifice plates makes each component solution gather bottom hole mixes well, by the reaction of mixed liquor obtained by S3
It is placed in incubation at room temperature 25-35 minutes；
S5,20ul fluorescein detection reagent is added in the every hole of white 96 orifice plates, then 96 orifice plates of white is placed on shaking table and are shaken
It shakes 2 minutes, 96 orifice plates of white is placed in room temperature later and continue to be incubated for 30 minutes；
S6, disk-read record the luminous value in every hole.
2. a kind of activity test method of deubiquitinating enzymes according to claim 1, it is characterised in that: the substrate is ammonia
Base fluorescein marks ubiquitin (Ub-AML), and the concentration of substrate is 0.6uM.
3. a kind of activity test method of deubiquitinating enzymes according to claim 1, it is characterised in that: contain in the S2
The 1X reaction buffer of 10mM DTT is diluted to obtain on the basis of being the reaction buffer after thawing in S1.
4. a kind of activity test method of deubiquitinating enzymes according to claim 1, it is characterised in that: the deubiquitination
Enzyme includes cysteine and all six other deubiquitination enzyme families of metalloproteinases.
5. a kind of activity test method of deubiquitinating enzymes according to claim 1, it is characterised in that: gone in the S2 general
Elementization enzyme working solution, which passes through respectively to pour into the 1X reaction buffer containing 10mM DTT with substrate working solution, to be filled
It is made in the glassware of ubiquitination enzyme-to-substrate.
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| CN201910806260.0A CN110412004A (en) | 2019-08-29 | 2019-08-29 | A kind of activity test method of deubiquitinating enzymes |
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| CN201910806260.0A CN110412004A (en) | 2019-08-29 | 2019-08-29 | A kind of activity test method of deubiquitinating enzymes |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989411A (en) * | 2004-06-21 | 2007-06-27 | 普罗吉安拉公司 | Diagnostic and screening methods and kits associated with proteolytic activity |
| US20070166778A1 (en) * | 2006-01-13 | 2007-07-19 | Xavier Jacq | Substrates and methods for assaying deubiquitinating enzymes |
| WO2009058730A1 (en) * | 2007-10-29 | 2009-05-07 | Schering Corporation | Diamido thiazole derivatives as protein kinase inhibitors |
| US20120058499A1 (en) * | 2010-06-11 | 2012-03-08 | Orcutt Steven J | Bioluminescent Detection of Protease Activity |
| US20130295595A1 (en) * | 2012-05-02 | 2013-11-07 | Katerina Heran Darwin | Modified prokaryotic ubiquitin-like protein and methods of use thereof |
| CN104844704A (en) * | 2014-02-17 | 2015-08-19 | 华东师范大学 | Ub-Nanoluc reporter gene system and Ub-Ub-GS-Nanoluc reporter gene system, constructions and applications thereof |
| CN110172495A (en) * | 2019-06-05 | 2019-08-27 | 武汉合研生物医药科技有限公司 | A kind of rapid detection method of RSK4 enzymatic activity and its application |
-
2019
- 2019-08-29 CN CN201910806260.0A patent/CN110412004A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1989411A (en) * | 2004-06-21 | 2007-06-27 | 普罗吉安拉公司 | Diagnostic and screening methods and kits associated with proteolytic activity |
| US20070166778A1 (en) * | 2006-01-13 | 2007-07-19 | Xavier Jacq | Substrates and methods for assaying deubiquitinating enzymes |
| WO2009058730A1 (en) * | 2007-10-29 | 2009-05-07 | Schering Corporation | Diamido thiazole derivatives as protein kinase inhibitors |
| US20120058499A1 (en) * | 2010-06-11 | 2012-03-08 | Orcutt Steven J | Bioluminescent Detection of Protease Activity |
| US20130295595A1 (en) * | 2012-05-02 | 2013-11-07 | Katerina Heran Darwin | Modified prokaryotic ubiquitin-like protein and methods of use thereof |
| CN104844704A (en) * | 2014-02-17 | 2015-08-19 | 华东师范大学 | Ub-Nanoluc reporter gene system and Ub-Ub-GS-Nanoluc reporter gene system, constructions and applications thereof |
| CN110172495A (en) * | 2019-06-05 | 2019-08-27 | 武汉合研生物医药科技有限公司 | A kind of rapid detection method of RSK4 enzymatic activity and its application |
Non-Patent Citations (1)
| Title |
|---|
| STEVEN J.ORCUTT ET AL.: "Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
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Application publication date: 20191105 |
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