CN110172495A - A kind of rapid detection method of RSK4 enzymatic activity and its application - Google Patents
A kind of rapid detection method of RSK4 enzymatic activity and its application Download PDFInfo
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- CN110172495A CN110172495A CN201910486214.7A CN201910486214A CN110172495A CN 110172495 A CN110172495 A CN 110172495A CN 201910486214 A CN201910486214 A CN 201910486214A CN 110172495 A CN110172495 A CN 110172495A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
Abstract
The present invention relates to a kind of rapid detection method of RSK4 enzymatic activity and its applications.Detection method are as follows: (1) take sample to be tested, blank control, positive control to be incubated for altogether with RSK4 enzyme, RSK peptide substrate/ATP mixed liquor respectively and carry out kinase reaction generation ADP；(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, is terminated kinase reaction and is run out of remaining ATP；(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, ADP is made to be converted to ATP, and read the luminous value RLU of newly synthesized ATP；(4) enzyme activity is calculated by enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%.This method is based on ADP-GloTMKinase assay, the ATP directly consumed in measurement reaction carry out the process of quantitative reaction.This method operates under the conditions of common lab, avoids causing radiation injury to operator and environment；And stable system, determination data accuracy is high, can be applied to the screening of RSK4 target spot inhibitor and the screening of RSK4 target spot anti-tumor drug.
Description
Technical field
The invention belongs to technological field of biochemistry, and in particular to a kind of rapid detection method of RSK4 enzymatic activity and its answer
With.
Background technique
RSK4 albumen size is 90-kDa, is ribosome S 6 kinase enzyme family important member, belongs to the increasing for adjusting cell growth
The growth factor grow, survive, broken up.RSK4 is activated after ERK and PDK1 phosphorylation.RSK4 usually with continuous gene syndrome
It is related, including x linked deaf (dfn3), baryencephalia (mrx) and choroideremia disease (chm).RSK4 is under normal circumstances in brain
Portion and the expression of kidney height, and in colorectal cancer patients, RSK4 expression quantity significantly increases.In the patient of non-small cell lung cancer,
RSK4 content is substantially less than normal tissue in cancerous issue, and RSK4 constitutes a hypothesis in clinical pilot studies data
NSCLC tumor suppressor gene.
The evaluation method of building isotope is generallyd use in previous Research Literature to measure the enzymatic activity of RSK4.This side
Method is made marks using 32P.32P half-life period has 14.3 days, and β ray is released in decay.Isotope experiment is needed in dedicated isotope
It is carried out in laboratory, and is equipped with corresponding protective shielding, gauge check instrument and necessary Emergency Tool are set.It is engaged in radiation work
The personnel of work must have corresponding profession and Protection Knowledge and healthiness condition, and provide corresponding testimonial material and staff
Health account.Staff is equipped with the personal protections such as dedicated work clothes, shoes, cap, mask, oversleeve, gloves, breathing mask and uses
Product.Laboratory hardware and qualification are required using the compound activity evaluation that isotope method carries out RSK4, limit experiment
Development.And isotope reagent price is expensive, reagent treatment cost of waste liquor is high, is not suitable as Large-scale Screening experimental evaluation chemical combination
The activity in object library.
Summary of the invention
For technological gap in the prior art, the object of the present invention is to provide a kind of quick detection sides of RSK4 enzymatic activity
Method and its application, this method utilize RSK4 albumen can be by the principle of substrate phosphorylation, in conjunction with ADP-Glo in molecular levelTMThis
The amount of the kind very high technology of sensitivity, the ATP directly consumed in measurement reaction carrys out the process of quantitative reaction.Entire reaction is general
It is operated under logical laboratory condition, avoids the radiation risk that may cause to operator and environment；The same day achievable operation, greatly
Detection time is shortened greatly.The screening that this method can be applied to the inhibitor of RSK4 kinases target spot is relevant anti-with for this target spot
The screening of tumour medicine.
The present invention is achieved by the following technical solutions:
The first purpose of the invention is to provide a kind of rapid detection methods of RSK4 enzymatic activity, comprising the following steps:
(1) take sample to be tested, blank control product, positive reference substance mixed with RSK4 enzymatic reagent, RSK peptide substrate/ATP respectively
It closes liquid and is incubated for progress kinase reaction generation ADP altogether；
(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, is terminated and is swashed
Enzyme reaction simultaneously runs out of remaining ATP；
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, be converted to ADP
ATP, and read the luminous value RLU of newly synthesized ATP；
(4) enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100% is pressed
Calculate the activity of RSK4 enzyme；Wherein RLU (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, RLU
(Blank) to be not added with RSK4 enzyme in sample to be tested kinases readings when.
Further, the sample to be tested of the step (1) is dissolved in buffer for untested compound and is formed by solution, sun
Property reference substance be that general kinase inhibitor staurosporine is dissolved in buffer and is formed by solution, blank control is buffer, institute
Stating buffer includes 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer 7.5.
Further, RSK4 enzymatic reagent concentration is 0.25-2.5ng/ μ l in the step (1), and sample to be tested concentration is 50 μ
M-0.64nM, positive reference substance concentration are 2 μM of -0.0256nM, RSK peptide substrate concentration in RSK peptide substrate/ATP mixed liquor
For 0.5mg/ml, ATP concentration is 62.5 μM.
Further, the total incubation temperature of the step (1) is 25-30 DEG C, and incubation time is 30-120min altogether.
Further, the ADP-Glo of the step (2)TMReaction reagent and the kinase reaction system of step (1) are isometric.
Further, the total incubation temperature of the step (2) is 25-30 DEG C, and incubation time is 30-60min altogether.
A second object of the present invention is to provide the rapid detection methods of RSK4 enzymatic activity as described in any one of the above embodiments to exist
Application in the screening of RSK4 target spot inhibitor.
Third object of the present invention is to provide the rapid detection methods of RSK4 enzymatic activity as described in any one of the above embodiments to exist
Application in the screening of RSK4 target spot anti-tumor drug.
Fourth object of the present invention is to provide a kind of screening reagent box of RSK4 target spot inhibitor, comprising: buffer, sky
White reference substance, positive reference substance, RSK4 enzymatic reagent, RSK peptide substrate/ATP mixed liquor and ADP-GloTMReaction reagent and kinases
Detection reagent, the buffer include 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer=
7.5；The blank control product are buffer；The positive reference substance is that general kinase inhibitor staurosporine is dissolved in buffer
It is formed by solution, concentration is 2 μM of -0.0256nM；The RSK4 enzymatic reagent is dissolved in buffer for RSK4 enzyme and is formed by
Solution, concentration are 0.25-2.5ng/ μ l；RSK peptide substrate/ATP the mixed liquor is dissolved in slow for ATP and RSK peptide substrate
Fliud flushing is formed by solution, and ATP concentration is 62.5 μM, and RSK peptide substrate concentration is 0.5mg/ml.
Using the method for the screening reagent box screening RSK4 target spot inhibitor of above-mentioned RSK4 target spot inhibitor, comprising:
(1) each 1 μ l of sample to be tested, blank control product, positive reference substance is taken, three different holes of microwell plate are added to
In, microwell plate centrifugation makes untested compound and positive control gather microwell plate bottom；
(2) 2 μ l RSK4 enzymatic reagents are respectively added into three micropores of step (1), add rear microwell plate centrifugation；
(3) 2 μ l RSK peptide substrates/ATP mixed liquor is respectively added into three micropores of step (2), adds rear microwell plate
Centrifugation；
(4) after being centrifuged, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 30-120min altogether under the conditions of 25-30 DEG C；
(5) step (4) terminates after being incubated for, and takes ADP-GloTM5 hole μ l/ of reaction reagent is added separately in three micropores, micro-
Orifice plate is centrifuged, and is incubated for 30-60min under the conditions of 25-30 DEG C；
(6) step (5) terminates after being incubated for, and takes 10 hole μ l/ of kinase assay reagent to be added separately in three micropores, microwell plate
It is centrifuged, is incubated for 20-60 minutes under the conditions of 25-30 DEG C；
(7) step (6) terminates after being incubated for, and carries out chemiluminescence detection in plate reader, reads luminous value (RLU), by with
Lower formula calculates enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%；Wherein
RLU (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, and RLU (Blank) is not add in sample to be tested
Kinases readings when adding RSK4 enzyme.
Compared with prior art, the advantages of the present invention are as follows:
(1) rapid detection method of RSK4 enzymatic activity provided by the invention utilizes RSK4 albumen can be the bottom of by molecular level
The principle of object phosphorylation, in conjunction with ADP-GloTMThis very high technology of sensitivity, the amount of the ATP directly consumed in measurement reaction
Carry out the process of quantitative reaction.Experiment shows this method stable system, and determination data accuracy is high.Therefore, this method can be applied to
The screening of RSK4 target spot inhibitor and the screening of RSK4 target spot anti-tumor drug.
(2) rapid detection method of RSK4 enzymatic activity of the invention can operate under the conditions of common lab, not need
Isotopic laboratory condition reduces the radiation risk that may cause to operator and environment.Operation is simple for this method,
The same day can complete to operate, and substantially reduce sample detection time, improve detection efficiency.
(3) method of the invention carries out reaction in 384 orifice plates, and system is 20 μ l systems, the reality with general 96 orifice plate
It tests compared to test volume is reduced, it is real to be particularly suited for a large amount of compound activity evaluation for the data volume of the single detection of raising
It tests.
(4) Z factor of detection architecture of the invention is 0.63, shows that system of the present invention is suitable for high flux screening.
Detailed description of the invention
Fig. 1 is test of the general kinase inhibitor staurosporine (staurosporine) in the kinases system of embodiment 1
As a result；Figure 1A, 1B, 1C are respectively to measure test value of the staurosporine in the kinases system for the first time, for the second time, for the third time.
Fig. 2 is the homogeneity evaluation result schematic diagram of detection architecture of the invention.
Fig. 3 is the test result in the kinases system of BI-D1870 in the present invention.
Specific embodiment
Applicant is in conjunction with specific embodiments described in further details technical solution of the present invention below, so that this field
Technical staff may be better understood the present invention and can be practiced, but range is claimed not as the present invention in illustrated embodiment
Restriction.
The rapid detection method of embodiment 1:RSK4 enzymatic activity
In the present embodiment, RSK4 recombinant protein, RSK peptide substrate, 5X polypeptide buffer and DTT are purchased from Signalchem,
ADP-GloTMKinase reagent is purchased from Promega.
1, a kind of rapid detection method of RSK4 enzymatic activity, comprising the following steps:
(1) take sample to be tested, blank control product, positive reference substance mixed with RSK4 enzymatic reagent, RSK peptide substrate/ATP respectively
It closes liquid and is incubated for progress kinase reaction generation ADP altogether；Sample to be tested, positive reference substance, RSK4 enzymatic reagent, RSK peptide substrate/ATP
Mixed liquor is using buffer as solvent, and wherein positive reference substance is dissolved in buffer institute shape for general kinase inhibitor staurosporine
At solution, concentration be 2 μM of -0.0256nM；RSK4 enzymatic reagent concentration be 0.25-2.5ng/ μ l, sample to be tested concentration be 50 μM-
RSK peptide substrate concentration is 0.5mg/ml in 0.64nM, RSK peptide substrate/ATP mixed liquor, and ATP concentration is 62.5 μM.Blank
Control is buffer.In the present embodiment, buffer includes 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM
DTT, pH of buffer 7.5.It is 25-30 DEG C that the reaction system, which is total to incubation temperature, and incubation time is 30-120min altogether.
(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, is terminated and is swashed
Enzyme reaction simultaneously runs out of remaining ATP；ADP-GloTMReaction reagent and the kinase reaction system of step (1) are isometric, are incubated for temperature altogether
Degree is 25-30 DEG C, and incubation time is 30-60min altogether.
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, be converted to ADP
ATP, and read the luminous value RLU of newly synthesized ATP；It is 25-30 DEG C that the reaction system, which is total to incubation temperature, and incubation time is altogether
20-60min。
(4) enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100% is pressed
Calculate the activity of RSK4 enzyme；Wherein RLU (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, RLU
(Blank) to be not added with RSK4 enzyme in sample to be tested kinases readings when.
2, the rapid detection method of RSK4 enzymatic activity of the invention can be applied to screening and the RSK4 of RSK4 target spot inhibitor
The screening of target spot anti-tumor drug.
3, a kind of screening reagent box of RSK4 target spot inhibitor, comprising: buffer, blank control product, positive reference substance,
RSK4 enzymatic reagent, RSK peptide substrate/ATP mixed liquor, ADP-GloTMReaction reagent and kinase assay reagent, the buffer packet
Include 40mM Tris, 20mM MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer=7.5；The blank control product are
Buffer；The positive reference substance is dissolved in buffer for general kinase inhibitor staurosporine and is formed by solution, concentration
For 2 μM of -0.0256nM；The RSK4 enzymatic reagent is dissolved in buffer for RSK4 enzyme and is formed by solution, concentration 0.25-
2.5ng/μl；RSK peptide substrate/ATP the mixed liquor is dissolved in buffer for ATP and RSK peptide substrate and is formed by solution,
ATP concentration is 62.5 μM, and RSK peptide substrate concentration is 0.5mg/ml.
4, using the method for the screening reagent box screening RSK4 target spot inhibitor of above-mentioned RSK4 target spot inhibitor, step is such as
Under:
Step 1:
(1) defrosting RSK4 enzyme, RSK peptide substrate, 5X buffer (200mM Tris on ice；pH 7.5；100mM
MgCl2；0.5mg/ml BSA) and DTT (0.1M), and the above reagent needs to be placed on ice always in the entire experiment process
On；ADP-GloTMATP in kinase reagent is also required to dissolve on ice, and needs to be placed on always in the entire experiment process
On ice.
(2) buffer of 5X is diluted to 1X with deionized water, and DTT is added wherein, being made into DTT concentration is 50 μM
1X buffer.
(3) it prepares sample to be tested: sample to be tested being taken to be dissolved with DMSO, the DMSO concentration of compound cannot be greater than 5%, experiment
Final DMSO concentration cannot be greater than 1%；Then testing sample solution is further diluted with 1X buffer, makes 50 μM of its concentration-
0.64nM。
(4) it prepares positive reference substance: staurosporine being taken to be dissolved with DMSO, the DMSO concentration of staurosporine cannot be greater than 5%,
1% cannot be greater than by testing final DMSO concentration；Then staurosporine solution is further diluted with 1X buffer, makes 2 μ of its concentration
M-0.0256nM。
(5) take each 1 μ l of sample to be tested, blank control product, positive reference substance, be added to three of white microwell plate it is different
Kong Zhong, microwell plate are centrifuged 1 minute for 1000 turns on centrifuge, and untested compound and positive control is made to gather microwell plate bottom.
Wherein blank control is 1X buffer.
Step 2,
(1) after RSK4 enzyme thaws completely, RSK4 enzyme is diluted to 0.5ng/ μ l using the buffer of 1X.
(2) 2 μ l RSK4 enzymatic reagents are respectively added into three micropores in step 1, the enzyme amount of RSK4 enzyme is in every hole at this time
1ng；2 hole μ l/ 1X buffers are added in blank control wells；This step carries out on ice, and after adding, microwell plate is 1000 on centrifuge
Turn centrifugation 1 minute.
Step 3,
(1) it configures RSK peptide substrate/ATP mixed liquor: using 1X buffer, be made into ATP for 160 times of 10mM ATP dilution and delay
Fliud flushing；RSK peptide substrate (1mg/ml) is subjected to 2 times of dilutions with ATP buffer and is made into RSK peptide substrate/ATP mixed liquor, this
When ATP concentration be 62.5uM, RSK peptide substrate concentration be 0.5mg/ml.This step carries out on ice.
(2) 2 μ l RSK peptide substrates/ATP mixed liquor is respectively added into three micropores of step 2, adds rear microwell plate
1000 turns are centrifuged 1 minute.
After step 4, centrifugation, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 30-120min altogether under the conditions of 25-30 DEG C；
Step 5,
(1) the ADP-GloTM reaction reagent of needs and kinase assay related reagent are equilibrated into room temperature, by 10mL kinases
Detection buffer is added to mixing for standby use in kinase assay substrate dry powder.Extra reagent dispenses -20 DEG C of preservations.
(2) after the reaction system of step 4 terminates incubation, ADP-Glo is takenTM5 hole μ l/ of reaction reagent be added separately to three it is micro-
Kong Zhong, microwell plate are centrifuged, and are incubated for 30-60min under the conditions of 25-30 DEG C.
Step 6,
Step (5) terminates after being incubated for, and takes 10 hole μ l/ of kinase assay reagent to be added separately in three micropores, microwell plate
1000 turns are centrifuged 1 minute, are incubated for 20-60 minutes under the conditions of 25-30 DEG C；
Step 7,
Step 6 terminates after being incubated for, and chemiluminescence detection is carried out in plate reader, reads luminous value (RLU), as follows
Calculate enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%, wherein
RLU (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, and RLU (Blank) is not add in sample to be tested
Kinases readings when adding RSK4 enzyme.
5, the signal of differential responses enzyme amount is than measurement
According to above-mentioned reaction system, different reaction enzyme amount is added during the reaction, tests signal-to-noise ratio.It was found that when enzyme is used
When amount is 0.5-5ng/ reaction, signal-to-noise ratio is greater than 5 times or more, is all satisfied initial reaction requirement.Using 1ng/ reaction as subsequent
The reaction condition of experiment.
The signal of 1 differential responses enzyme amount of table compares measurement result
6, the signal of differential responses duration is than measurement
When enzyme dosage is fixed as the every reaction of 1ng, using different kinase reaction incubation times, from 30 minutes to 120 point
Clock gradually extends the reaction time.As a result, it has been found that when incubation time is from 30 minutes to 120 minute, obtained Signal-to-Noise by
It is cumulative to add, and it is all satisfied greater than 5 times signal testing requirements.Subsequent experimental uses the every reaction of 1ng, does as the reaction time within 60 minutes
Downstream time.
The signal of 2 differential responses duration of table compares measurement result
| Average chemical shines readings | Background average chemical shines readings | Signal-to-noise ratio | |
| 30 minutes | 5429 | 870 | 6.24 |
| 60 minutes | 28488 | 1450 | 19.64 |
| 90 minutes | 45928 | 2190 | 20.97 |
| 120 minutes | 68903 | 2518 | 27.36 |
7, positive reference compound determination
It is 60 minutes when the kinase reaction time, when reaction enzyme amount is 1ng, measures general kinase inhibitor staurosporine
(staurosporine) test value in this kinases system, the positive reference index as system.As shown in Figure 1, independent inspection
Survey half-inhibitory concentration of the staurosporine in system, respectively 1.6nM, 1.1nM and 1.2nM three times.Parallel laboratory test knot three times
Fruit determination data difference is within three times, it is believed that determination data accuracy is higher, and experimental system is stablized.Determination data can be used as
The positive reference index of subsequent experimental
8, system homogeneity is evaluated
The Z- factor (Z-factor) is a combination about signal spacing and variation, it has become assessment test method matter
The major parameter of amount.The Z- factor is the measurement of Statistical Effect size, its proposition is for judging to measure in high flux screening
Whether response intensity reaches the requirement further studied.The value of the Z- factor is one for distinguishing the opposite of signal and background population
Index.It is a parameter that do not measure, and range can be from " 1 to less than 0 ".When the Z- factor is equal to zero, signal and back
Scape starts to be overlapped.In general, the acceptable Z- factor should be greater than 0.4.
We measure the Z factor index of signal and background in the present invention.Measuring Z factor is 0.63, illustrates body of the present invention
System is suitable for high flux screening.
Embodiment 2: detection method of the invention carries out the screening of RSK4 target spot inhibitor to BI-D1870
It takes BI-D1870 to test according to the method for embodiment 1, and calculates its enzyme activity and IC50 value to RSK4, such as Fig. 3 institute
Show, the IC50 of RSK4 is 24.3nM.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of rapid detection method of RSK4 enzymatic activity, which comprises the following steps:
(1) take sample to be tested, blank control product, positive reference substance respectively with RSK4 enzymatic reagent, RSK peptide substrate/ATP mixed liquor
It is incubated for altogether and carries out kinase reaction generation ADP；
(2) ADP-Glo is added respectively into three groups of kinase reaction systems of step (1)TMReaction reagent is incubated for altogether, and it is anti-to terminate kinases
It should and run out of remaining ATP；
(3) kinase assay reagent is added respectively into three groups of reaction systems of step (2) to be incubated for altogether, ADP is made to be converted to ATP, and
Read the luminous value RLU of newly synthesized ATP；
(4) it is calculated by enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%
The activity of RSK4 enzyme；Wherein RLU (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, RLU (Blank)
Kinases readings when to be not added with RSK4 enzyme in sample to be tested.
2. a kind of rapid detection method of RSK4 enzymatic activity according to claim 1, which is characterized in that the step (1)
Sample to be tested be that untested compound is dissolved in buffer and is formed by solution, positive reference substance is general kinase inhibitor star spore
Rhzomorph is dissolved in buffer and is formed by solution, and blank control is buffer, and the buffer includes 40mM Tris, 20mM
MgCl2, 0.1mg/ml BSA and 50 μM of DTT, pH of buffer 7.5.
3. a kind of rapid detection method of RSK4 enzymatic activity according to claim 1, which is characterized in that the step (1)
Middle RSK4 enzymatic reagent concentration is 0.25-2.5ng/ μ l, and sample to be tested concentration is 50 μM of -0.64nM, and positive reference substance concentration is 2 μ
RSK peptide substrate concentration is 0.5mg/ml in M-0.0256nM, RSK peptide substrate/ATP mixed liquor, and ATP concentration is 62.5 μM.
4. a kind of rapid detection method of RSK4 enzymatic activity according to claim 1, which is characterized in that the step (1)
Total incubation temperature be 25-30 DEG C, altogether incubation time be 30-120min.
5. a kind of rapid detection method of RSK4 enzymatic activity according to claim 1, which is characterized in that the step (2)
ADP-GloTMReaction reagent and the kinase reaction system of step (1) are isometric.
6. a kind of rapid detection method of RSK4 enzymatic activity according to claim 1, which is characterized in that the step (2)
Total incubation temperature be 25-30 DEG C, altogether incubation time be 30-60min.
7. the rapid detection method of RSK4 enzymatic activity as claimed in any one of claims 1 to 6 is in the screening of RSK4 target spot inhibitor
In application.
8. the rapid detection method of RSK4 enzymatic activity as claimed in any one of claims 1 to 6 is in RSK4 target spot anti-tumor drug
Application in screening.
9. a kind of screening reagent box of RSK4 target spot inhibitor characterized by comprising buffer, blank control product, the positive are right
It is described slow according to product, RSK4 enzymatic reagent, RSK peptide substrate/ATP mixed liquor, ADP-GloTM reaction reagent and kinase assay reagent
Fliud flushing includes 40mM Tris, 20mM MgCl2,0.1mg/ml BSA and 50 μM of DTT, pH of buffer=7.5；The blank pair
It is buffer according to product；The positive reference substance is dissolved in buffer for general kinase inhibitor staurosporine and is formed by solution,
Its concentration is 2 μM of -0.0256nM；The RSK4 enzymatic reagent is dissolved in buffer for RSK4 enzyme and is formed by solution, and concentration is
0.25-2.5ng/μl；RSK peptide substrate/ATP the mixed liquor is dissolved in buffer for ATP and RSK peptide substrate and is formed by
Solution, ATP concentration are 62.5 μM, and RSK peptide substrate concentration is 0.5mg/ml.
10. the method for the screening reagent box screening RSK4 target spot inhibitor of application RSK4 target spot inhibitor as claimed in claim 9,
It is characterised by comprising:
(1) each 1 μ l of sample to be tested, blank control product, positive reference substance is taken, is added in three different holes of microwell plate, it is micro-
Orifice plate centrifugation, makes untested compound and positive control gather microwell plate bottom；
(2) 2 μ l RSK4 enzymatic reagents are respectively added into three micropores of step (1), add rear microwell plate centrifugation；
(3) 2 μ l RSK peptide substrates/ATP mixed liquor is respectively added into three micropores of step (2), adds rear microwell plate centrifugation；
(4) after being centrifuged, microwell plate pad pasting is given, pad pasting is compressed, is incubated for 30-120min altogether under the conditions of 25-30 DEG C；
(5) step (4) terminates after being incubated for, and takes ADP-GloTM5 hole μ l/ of reaction reagent is added separately in three micropores, microwell plate
It is centrifuged, is incubated for 30-60min under the conditions of 25-30 DEG C；
(6) step (5) terminate be incubated for after, take 10 hole μ l/ of kinase assay reagent to be added separately in three micropores, microwell plate from
The heart is incubated for 20-60 minutes under the conditions of 25-30 DEG C；
(7) step (6) terminates after being incubated for, and chemiluminescence detection is carried out in plate reader, reads luminous value (RLU), by following public affairs
Formula calculates enzymatic activity:
Enzyme activity=(RLU (Sample)-RLU (Blank))/(RLU (Pos.Ctrl)-RLU (Blank)) × 100%；Wherein RLU
It (Pos.Ctrl) is the kinases readings of blank control product and RSK4 kinase reaction, RLU (Blank) is to be not added in sample to be tested
Kinases readings when RSK4 enzyme.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110412004A (en) * | 2019-08-29 | 2019-11-05 | 苏州新格诺康生物技术有限公司 | A kind of activity test method of deubiquitinating enzymes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110412004A (en) * | 2019-08-29 | 2019-11-05 | 苏州新格诺康生物技术有限公司 | A kind of activity test method of deubiquitinating enzymes |
| CN112362640A (en) * | 2020-10-11 | 2021-02-12 | 华中农业大学 | Screening method and application of streptococcus suis c-di-AMP synthetase inhibitor |
| CN112362640B (en) * | 2020-10-11 | 2021-10-26 | 华中农业大学 | Screening method and application of streptococcus suis c-di-AMP synthetase inhibitor |
| CN115436500A (en) * | 2021-06-04 | 2022-12-06 | 成都先导药物开发股份有限公司 | Method for identifying operable cut DNA coding seedling head compound |
| CN115969855A (en) * | 2022-12-15 | 2023-04-18 | 中国人民解放军空军军医大学 | Application of APY29 in preparation of RSK4 kinase inhibitor |
| CN115969855B (en) * | 2022-12-15 | 2024-01-26 | 中国人民解放军空军军医大学 | Application of APY29 in preparation of RSK4 kinase inhibitor |
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