A method for high-throughput screening of protein kinase a inhibitors or activators based on homogeneous chemiluminescence detection

Abstract

The present application relates to a kind of protein kinase A inhibitor or activator high-throughput screening method based on homogeneous chemiluminescence detection, belong to biological detection technical field.The existing protein kinase A (PKA) activity detection technique is tedious, and because PKA substrate is short peptide, it will seriously affect its combination with antibody when being fixed in high-throughput screening plate, thereby leading to the sensitivity of detection method to decline, cannot realize high-throughput screening.The present application first designs a new PKA substrate polypeptide, its structure is completely different from existing substrate polypeptide, and the binding efficiency of substrate phosphorylation with anti-phosphorylation antibody is higher.Therefore, the present application constructs a kind of protein kinase A detection system based on the element, and combines homogeneous chemiluminescence detection technology to realize high-throughput detection, simplifies detection system, improves the sensitivity of detection, and is verified in the screening of protein kinase A inhibitor or activator, and is expected to become a new PKA drug screening platform.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a method for high-throughput screening of protein kinase A inhibitors or activators based on homogeneous chemiluminescence detection. Background Technology

[0002] Protein kinases (PKs) are phosphotransferases responsible for transferring γ-phosphate from ATP to specific amino acid residues in substrate proteins. This process, known as protein phosphorylation, plays a crucial role in multiple biological processes, including cell cycle, growth, apoptosis, and signal transduction. Protein kinase A (PKA), also known as cAMP-dependent protein kinase, is an important member of the kinase family and a second messenger-dependent enzyme. It regulates PKA activity by influencing the activities of adenylate cyclase (AC) and phosphodiesterase, thereby determining cAMP levels and ultimately regulating cellular responses to various external stimuli, including cell growth, metabolism, DNA replication, cell division, and actin cytoskeleton rearrangement. PKA consists of two catalytic subunits and two regulatory subunits, existing as an inactive complex in the absence of cAMP. When cAMP binds to the regulatory subunits, it alters the conformation of the regulatory subunits, causing dissociation of the regulatory and catalytic subunits and releasing the catalytic subunit. Activated protein kinase A catalytic subunits can phosphorylate serine or threonine residues of certain proteins in the cell, thereby altering the activity of these proteins and further affecting the expression of related genes.

[0003] Homogeneous chemiluminescence detection technology is a novel chemiluminescence analysis technique developed based on LOCI (Luminescent Oxygen Channeling Assay) technology. Its principle is based on two types of microspheres: a donor and an acceptor. The donor microspheres contain photosensitizers (such as phthalocyanine molecules), which can generate singlet oxygen under specific wavelength light irradiation. The acceptor microspheres contain various compounds (such as dimethylthiophene and europium), which can react with singlet oxygen. When the donor microspheres are excited by 680 nm light, the photosensitizer releases singlet oxygen. Since singlet oxygen travels approximately 200 nm in water, it can only diffuse to the acceptor microspheres when the donor and acceptor microspheres are connected by an analyte (such as an antigen). At this point, the fluorescent substance in the acceptor microspheres can rapidly react with the singlet oxygen generated near the donor microspheres, exciting europium through energy transfer to emit a fluorescence signal at approximately 610 nm. Qualitative or quantitative detection can be achieved by detecting the intensity of this light signal.

[0004] Currently, most methods for detecting active PKA utilize the PKA activity in the sample to phosphorylate the PKA substrate coated on a 96-well plate, followed by ELISA detection using a specific antibody against the phosphorylated substrate. However, this method is cumbersome, time-consuming, and labor-intensive, making high-throughput screening difficult. Therefore, the development of convenient, rapid, and accurate detection methods for screening inhibitors or activators of PKA activity is receiving increasing attention in in vitro functional drug screening. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention combines homogeneous chemiluminescence detection technology and antibody detection technology, and optimizes the phosphorylated substrates and antibodies in the detection system to construct a method for high-throughput screening of protein kinase A inhibitors or activators. This method requires no antigen / antibody coating, no cumbersome plate washing steps, and is simple to prepare and detect, showing great potential in biomedical screening.

[0006] The first objective of this invention is to provide a method for high-throughput screening of protein kinase A inhibitors or activators, comprising the following steps:

[0007] S1. Conjugate a vector containing a LOCI donor or a vector containing a LOCI receptor to an anti-Flag antibody to obtain the first conjugate.

[0008] A second conjugate is obtained by conjugating a vector containing a LOCI receptor or a vector containing a LOCI donor with an antiphosphorylated substrate antibody; the first conjugate and the second conjugate may not contain a LOCI donor or may not contain a LOCI receptor simultaneously.

[0009] S2. Co-incubate the protein kinase A substrate with the first conjugate, protein kinase A, and ATP. After incubation, mix with the second conjugate and continue incubation. After incubation, detect the light signal intensity.

[0010] S3. Repeat step S2 and add the protein kinase A inhibitor or activator to be screened to the co-incubation system. Determine the inhibition or activation effect based on the change in light signal intensity to achieve high-throughput screening of protein kinase A inhibitors or activators.

[0011] The amino acid sequence of the protein kinase A substrate is shown in SEQ ID NO.1, and the antiphosphorylation substrate antibody specifically binds to the phosphorylated protein kinase A substrate.

[0012] Furthermore, different inhibitors and activators were added to different wells of the culture plate suitable for high-throughput screening to facilitate comparison of the light signal intensity with the blank control.

[0013] Furthermore, the antiphosphorylated substrate antibody is an antibody that specifically recognizes LRRAPSLG.

[0014] Furthermore, the carrier includes, but is not limited to, microspheres. The donor microspheres are energy donors that generate singlet oxygen upon photoexcitation and contain photosensitizers; the acceptor microspheres are energy acceptors and signal generators that receive singlet oxygen and convert it into a detectable light signal, and contain dyes.

[0015] Furthermore, when detecting the intensity of the light signal, the excitation wavelength is the wavelength range that can convert oxygen molecules in the environment surrounding the LOCI donor into singlet oxygen, such as 680 nm, and the detection wavelength is the wavelength range of the light signal generated after the LOCI acceptor receives singlet oxygen energy, such as 340-620 nm.

[0016] Furthermore, the anti-Flag antibody is a rabbit monoclonal antibody.

[0017] Furthermore, in the detection system, the mass ratio of protein kinase A substrate, first conjugate, protein kinase A, ATP, and second conjugate is (2-4): (60-120): 1: (40-60): (60-120). Most preferably, the mass ratio is 3:100:1:50:100.

[0018] Furthermore, the buffer system in the detection system is a buffer solution containing Tris-HCl, KCl and MgCl2, with a pH of 6.5-8.

[0019] Furthermore, the concentrations of each substance in the buffer solution are: Tris-HCl 10-30 mM, KCl 40-60 mM, and MgCl 25-20 mM.

[0020] A second objective of this invention is to provide a detection system comprising:

[0021] A protein kinase A substrate, the amino acid sequence of which is shown in SEQ ID NO.1;

[0022] ATP;

[0023] Protein kinase A;

[0024] The first conjugate is a conjugate of a carrier containing a LOCI donor or a carrier containing a LOCI receptor and an anti-Flag antibody.

[0025] The second conjugate is a conjugate of a LOCI receptor-containing vector or a LOCI donor-containing vector with an antiphosphorylated substrate antibody, wherein the antiphosphorylated substrate antibody is an antibody that specifically recognizes LRRAPSLG. The first conjugate and the second conjugate do not simultaneously contain a LOCI donor or do not simultaneously contain a LOCI receptor.

[0026] A third objective of this invention is to provide a protein kinase A substrate suitable for homogeneous chemiluminescence detection methods, the amino acid sequence of which is shown in SEQ ID NO.1.

[0027] A fourth object of the present invention is to provide the use of the method, detection system, protein kinase A substrate or protein kinase A phosphorylated substrate and antibody pair in the preparation of products for detecting protein kinase A activity or in screening substances that affect protein kinase A activity.

[0028] Furthermore, substances that affect protein kinase A activity can be protein kinase A inhibitors, protein kinase A activators, etc.

[0029] The fifth objective of this invention is to provide a high-throughput detection method for protein kinase A mutants, comprising the following steps:

[0030] (1) The first conjugate is obtained by conjugating a vector containing a LOCI donor or a vector containing a LOCI receptor with an anti-Flag antibody;

[0031] A second conjugate is obtained by conjugating a vector containing a LOCI receptor or a vector containing a LOCI donor with an antiphosphorylated substrate antibody; the first conjugate and the second conjugate may not contain a LOCI donor or may not contain a LOCI receptor simultaneously.

[0032] (2) The protein kinase A substrate was co-incubated with the first conjugate, protein kinase A with known different activities, and ATP. After incubation, it was mixed with the second conjugate and incubated for a longer period. After incubation, the light signal intensity was detected.

[0033] (3) Based on the results of step (2), establish a coordinate system for protein kinase A activity and light signal intensity;

[0034] (4) Replace the known protein kinase A with the protein kinase A mutant to be tested, repeat step (2), substitute the light signal intensity into the coordinate system of step (3), and calculate the activity of the protein kinase A mutant to be tested.

[0035] Furthermore, if only qualitative analysis is required, the intensity of the light signal in the test group and the control group in the high-throughput panel can be compared. If quantitative analysis is required, a standard curve can be plotted, and the light signal intensity of the test group can be substituted into the calculation.

[0036] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0037] This invention designs a 25-amino acid PKA substrate polypeptide with a tag protein at one end for binding to donor microspheres coated with a tag protein antibody, and a PKA substrate sequence at the other end, which, upon phosphorylation, binds to receptor microspheres coated with a phosphorylated substrate antibody. This invention, through appropriate structural design, allows the substrate polypeptide to bind more flexibly to both the tag protein antibody and the phosphorylated substrate antibody in solution, improving detection sensitivity. Furthermore, this invention, combined with homogeneous chemiluminescence detection technology, eliminates the need for antigen / antibody coating, cumbersome plate washing, and TMB color development. The detection substance is simply added directly to a 96-well plate, incubated at 37°C for 30 min, and fluorescence values ​​can be directly detected using a microplate reader. The operation is extremely simple and convenient, making it suitable for high-throughput screening of PKA kinase candidate drugs (inhibitors or activators). Attached Figure Description

[0038] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0039] Figure 1 This is the standard curve for PKA activity detection. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0041] The solution involved in this invention is as follows:

[0042] The FDA approval of imatinib in 2001 marked a breakthrough in molecularly targeted cancer therapy, heralding the emergence of kinase inhibitors as a key drug class both in oncology and beyond. Currently, 71 small molecule kinase inhibitors (SMKIs) have been approved by the FDA, with another 16 approved by other regulatory agencies. Oncology is their primary application area, but one-third of SMKIs in clinical development are for indications outside of oncology, such as rheumatoid arthritis. Clinical trial information for SMKIs shows that approximately 110 novel kinases are currently being explored as targets. The approximately 45 targets of approved kinase inhibitors represent only about 30% of the human kinase group, indicating that there are still significant unexplored opportunities in this class of drugs.

[0043] In the process of SMKI drug discovery, detecting the impact of candidate drugs on activation activity is an essential step. Current kinase activity detection methods are cumbersome, time-consuming, labor-intensive, and difficult to implement for high-throughput screening. The purpose of this invention is to establish a simple, convenient, and accurate homogeneous chemiluminescence detection method suitable for high-throughput screening of protein kinase A drugs (inhibitors or activators), capable of completing drug detection within 30 minutes.

[0044] Existing PKA activity detection techniques involve coating a peptide PKA substrate onto a 96-well plate, then adding a standard or test sample with PKA activity and ATP to initiate PKA substrate phosphorylation. Subsequently, an ELISA method is used to detect the phosphorylated substrate content using an antibody that recognizes the phosphorylated substrate. Besides being cumbersome and time-consuming, PKA substrates, being short peptides, have low coating efficiency on 96-well plates. Furthermore, as short peptides, steric hindrance after immobilization on a 96-well plate can affect antibody binding, all of which limit the sensitivity of this detection method.

[0045] This invention designs a 25-amino acid PKA substrate polypeptide with a Flag tag at one end for binding to donor microspheres coated with Flag antibodies, and a PKA substrate sequence at the other end. Phosphorylation of the Flag tag allows it to bind to receptor microspheres coated with phosphorylated substrate antibodies. However, the close proximity of the two ends leads to low efficiency in binding to both antibodies simultaneously due to steric hindrance. Therefore, this invention adds two linker peptides (GGGGS), linking the Flag tag and PKA substrate to their respective ends. This allows the substrate polypeptide to bind more flexibly to both Flag and phosphorylated substrate antibodies in solution, improving detection sensitivity.

[0046] This invention employs homogeneous chemiluminescence detection technology, which eliminates the need for antigen / antibody coating, cumbersome plate washing steps, and TMB color development. Simply add the detection substance directly to a 96-well plate, incubate at 37°C for 30 minutes, and the fluorescence value can be directly detected using an ELISA reader. The operation is very simple and convenient, making it suitable for high-throughput screening of PKA kinase candidate drugs (inhibitors or activators).

[0047] The sequence involved in this invention is as follows:

[0048] PKA substrate (SEQ ID NO. 1): DYKDDDDKGGGGSGGGGSLRRASLG.

[0049] The phosphorylated substrate that the antiphosphorylated substrate antibody specifically binds to is LRRAPSLG, with serine at position 5 being phosphorylated.

[0050] Example

[0051] I. Experimental Materials

[0052] PKA kinase activity assay kit (Abcam, ab139435), PKA kinase (Sigma, P5511), ATP (Sigma, A1852), PKA inhibitor H-89 (Selleck, S1582), PKA activator 8-Bromo-cAMP SodiumSalt (Selleck, S7857). Peptide synthesis of PKA substrate (DYKDDDDK–GGGGS–GGGGS–LRRASLG) (GenScript). LOCI donor microspheres (carboxyl group) (Weixing Biotechnology), LOCI receptor microspheres (carboxyl group) (Weixing Biotechnology), Flag Tag recombinant rabbit monoclonal antibody (ABclonal, AE063), anti-phosphorylation substrate (LRRApSLG) antibody.

[0053] II. Experimental Procedure

[0054] 1. Conjugation of LOCI donor microspheres with Flag Tag recombinant rabbit monoclonal antibody

[0055] The coupling process between microspheres and antibodies is as follows:

[0056] 1) Cleaning: Sonicate the microspheres and place the required amount of microspheres into a centrifuge tube of appropriate volume. Add cleaning solution 1 (50mM MES) to the centrifuge tube, mix well, and then centrifuge for 15 minutes. Remove the supernatant. Add cleaning solution 1 again to the centrifuge tube, mix well, and then centrifuge for 15 minutes. Remove the supernatant.

[0057] 2) Activation: Add washing solution 1 (50mM MES) to the centrifuge tube at 1 times the original volume of the microspheres. Then add EDC solution and NHS solution, and activate at room temperature for 30 min.

[0058] 3) Washing: Add washing solution 2 (50mM PB pH=7) to the centrifuge tube, mix well, then centrifuge for 15 minutes and remove the supernatant. Add washing solution 2 again to the centrifuge tube, mix well, then centrifuge for 15 minutes and remove the supernatant. Add washing solution 2 again.

[0059] 4) Coupling: After activation, quickly add the antibody to the microsphere suspension and mix thoroughly. Place on a rotary instrument at 37°C in the dark for 1.0 h for coupling.

[0060] 5) Sealing: After coupling is complete, remove the centrifuge tube, sonicate it, add the sealing agent, and mix well. Place it on a rotary instrument at 37℃±2℃ in the dark for 2.0 h.

[0061] 6) Cleaning: After sealing, add cleaning solution 3 (0.1% Tween-20) to the centrifuge tube, mix well, then centrifuge for 15 minutes and remove the supernatant. Add cleaning solution 3 to the centrifuge tube again, mix well, then centrifuge for 15 minutes and remove the supernatant.

[0062] 7) Resuspension: Add 3 parts washing solution to the original volume of the microspheres in a centrifuge tube after removing the supernatant, and sonicate.

[0063] 8) Labeling: Affix a label to the outer wall of the container, indicating the product name, batch number, volume, preparation date, storage conditions, and usage period.

[0064] 2. LOCI receptor microspheres conjugated with anti-phosphorylation substrate antibodies

[0065] The coupling process between microspheres and antibodies is the same as in (1).

[0066] 3. Establishment of a homogeneous chemiluminescence detection method

[0067] 1) Prepare PKA substrates at a series of concentrations of 0 μg / ml, 1 μg / ml, 4 μg / ml, 16 μg / ml, 64 μg / ml, 128 μg / ml, 512 μg / ml, and 1024 μg / ml using standard buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2).

[0068] 2) The homogeneous chemiluminescence reaction was carried out in a black 96-well microplate. 10 μl of PKA substrate of different concentrations, 20 μl of donor microspheres coated with Flag antibody, 10 μl of PKA kinase (20 μg / ml), and 10 μl of ATP (1 mg / ml) were added sequentially, and the plate was incubated at 37°C for 15 min.

[0069] 3) Add 20 μl of receptor microspheres coated with phosphorylated substrate antibody and incubate at 37°C for 15 min.

[0070] 4) After incubation, the light signal intensity was detected using a multi-functional microplate reader (SpectraMax M5e, Molecular Devices) (excitation wavelength 680nm, detection wavelength 615nm).

[0071] 5) Based on the test results, select an appropriate PKA substrate concentration as a reference concentration for screening PKA activity inhibitors or activators.

[0072] 4. Screening of PKA activity inhibitors

[0073] 1) In a black 96-well microplate, add 10 μl of PKA substrate, 20 μl of donor microspheres coated with Flag antibody, 10 μl of PKA kinase, 10 μl of ATP (1 mg / ml), and different concentrations of PKA inhibitor H-89 in sequence, and incubate at 37°C for 15 min.

[0074] 2) Add 20 μl of receptor microspheres coated with phosphorylated substrate antibody and incubate at 37°C for 15 min.

[0075] 3) After incubation, the light signal intensity was detected using a multi-functional microplate reader (SpectraMax M5e, Molecular Devices) (excitation wavelength 680nm, detection wavelength 615nm).

[0076] 4) Based on the test results, adjust the concentrations of PKA substrate and PKA kinase so that the test results can fully reflect the inhibitory effect of H-89 on PKA kinase activity.

[0077] 5. Screening of PKA activators

[0078] 1) In a black 96-well microplate, add 10 μl of PKA substrate, 20 μl of donor microspheres coated with Flag antibody, 10 μl of PKA kinase, 10 μl of ATP (1 mg / ml), and different concentrations of PKA activator 8-Bromo-cAMP, and incubate at 37°C for 15 min.

[0079] 2) Add 20 μl of receptor microspheres coated with phosphorylated substrate antibody and incubate at 37°C for 15 min.

[0080] 3) After incubation, the light signal intensity was detected using a multi-functional microplate reader (SpectraMax M5e, Molecular Devices) (excitation wavelength 680nm, detection wavelength 615nm).

[0081] 4) Based on the test results, adjust the concentrations of PKA substrate and PKA kinase so that the test results can fully reflect the activation ability of 8-Bromo-cAMP on PKA kinase activity.

[0082] III. Experimental Results

[0083] This homogeneous chemiluminescence detection method can be applied to the detection of PKA activity. H-89 effectively inhibits PKA activity. With a fixed PKA concentration, as the concentration of the inhibitor H-89 gradually increases, the fluorescence signal of the system gradually decreases, indicating that increasing the concentration of H-89 gradually inhibits PKA activity, leading to a gradual decrease in phosphorylated peptide substrates and a gradual decrease in fluorescence intensity. Conversely, 8-Bromo-cAMP effectively activates PKA activity. With a fixed PKA concentration, as the concentration of the activator 8-Bromo-cAMP gradually increases, the fluorescence signal of the system gradually increases, indicating that increasing the concentration of 8-Bromo-cAMP gradually activates PKA activity, leading to a gradual increase in phosphorylated peptide substrates and a gradual increase in fluorescence intensity.

[0084] This invention constructs a homogeneous chemiluminescence detection method for quantitative detection of PKA activity. This method is simple and convenient to operate, provides accurate results, and can complete drug detection in 30 minutes, making it suitable for high-throughput screening of PKA kinase candidate drugs (inhibitors or activators).

[0085] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for high-throughput screening of protein kinase A inhibitors or activators, characterized in that, Includes the following steps: S1. Conjugate a vector containing a LOCI donor or a vector containing a LOCI receptor to an anti-Flag antibody to obtain the first conjugate. A second conjugate is obtained by conjugating a vector containing a LOCI receptor or a vector containing a LOCI donor with an antiphosphorylated substrate antibody; the first conjugate and the second conjugate may not contain a LOCI donor or may not contain a LOCI receptor simultaneously. S2. Co-incubate the protein kinase A substrate with the first conjugate, protein kinase A, and ATP. After incubation, mix with the second conjugate and continue incubation. After incubation, detect the light signal intensity. S3. Repeat step S2 and add the protein kinase A inhibitor or activator to be screened to the co-incubation system. Determine the inhibition or activation effect based on the change in light signal intensity to achieve high-throughput screening of protein kinase A inhibitors or activators. The amino acid sequence of the protein kinase A substrate is shown in SEQ ID NO.1, and the antiphosphorylation substrate antibody specifically binds to the phosphorylated protein kinase A substrate.

2. The method according to claim 1, characterized in that, The antiphosphorylated substrate antibody is an antibody that specifically recognizes LLRAPSLG.

3. The method according to claim 1 or 2, characterized in that, The mass ratio of protein kinase A substrate, first conjugate, protein kinase A, ATP, and second conjugate is (2-4): (60-120): 1: (40-60): (60-120).

4. The method according to claim 1 or 2, characterized in that, The carrier includes microspheres; And / or, when detecting the intensity of the light signal, the excitation wavelength is the wavelength range that can convert oxygen molecules in the environment surrounding the LOCI donor into singlet oxygen, and the detection wavelength is the wavelength range of the light signal generated after the LOCI acceptor receives singlet oxygen energy.

5. The method according to claim 1 or 2, characterized in that, The anti-Flag antibody is a rabbit monoclonal antibody; And / or, the buffer system in the detection system is a buffer solution containing Tris-HCl, KCl and MgCl2, pH 6.5-8.

6. A detection system, characterized in that, include: A protein kinase A substrate, the amino acid sequence of which is shown in SEQ ID NO.1; ATP; Protein kinase A; The first conjugate is a conjugate of a carrier containing a LOCI donor or a carrier containing a LOCI receptor and an anti-Flag antibody. The second conjugate is a conjugate of a LOCI receptor-containing vector or a LOCI donor-containing vector with an antiphosphorylated substrate antibody, wherein the antiphosphorylated substrate antibody is an antibody that specifically recognizes LRRAPSLG. The first conjugate and the second conjugate do not simultaneously contain a LOCI donor or do not simultaneously contain a LOCI receptor.

7. A protein kinase A substrate, characterized in that, The amino acid sequence of the protein kinase A substrate is shown in SEQ ID NO.

1.

8. The use of the method of any one of claims 1-5, the detection system of claim 6, or the protein kinase A substrate of claim 7 in the preparation of products for detecting protein kinase A activity or in screening substances that affect protein kinase A activity.

9. The application according to claim 8, characterized in that, The substances that affect protein kinase A activity include protein kinase A inhibitors or protein kinase A activators.

10. A method for detecting the activity of protein kinase A mutants, characterized in that, Includes the following steps: (1) The first conjugate is obtained by conjugating a vector containing a LOCI donor or a vector containing a LOCI receptor with an anti-Flag antibody; A second conjugate is obtained by conjugating a vector containing a LOCI receptor or a vector containing a LOCI donor with an antiphosphorylated substrate antibody; the first conjugate and the second conjugate may not contain a LOCI donor or may not contain a LOCI receptor simultaneously. (2) The protein kinase A substrate was co-incubated with the first conjugate, protein kinase A with known different activities, and ATP. After incubation, it was mixed with the second conjugate and incubated for a longer period. After incubation, the light signal intensity was detected. (3) Based on the results of step (2), establish a coordinate system for protein kinase A activity and light signal intensity; (4) Replace the known protein kinase A with the protein kinase A mutant to be tested, repeat step (2), substitute the light signal intensity into the coordinate system of step (3), and calculate the activity of the protein kinase A mutant to be tested.

Images