An elisa kit for quantitatively detecting s-palmitoylation modification target protein and a method of using the same

Abstract

The present application relates to the technical field of protein detection, and particularly relates to an ELISA detection kit for quantitatively detecting S-palmitoylation modified target proteins and a use method thereof; the kit comprises Protein G magnetic beads, a 96-well transparent polystyrene microwell plate, a 96-well plate magnetic frame, solution A, solution B, solution C, solution D, solution E, solution F, solution G; the present application relates to the comprehensive application of immunoprecipitation (IP), click chemistry and enzyme-linked immunosorbent (ELISA) signal amplification experimental principles, and realizes the effect of high-throughput quantitative detection of S-palmitoylation modified target proteins, is suitable for the mechanism and function research of S-palmitoylation modified target proteins of various samples (such as high-throughput screening of small molecule inhibitors targeting S-palmitoylation modified target proteins), and improves the experimental efficiency.

Description

Technical Field

[0001] This invention belongs to the field of protein detection technology, and specifically relates to an ELISA detection kit for quantitative detection of S-palmitoylated target proteins and its usage method. Background Technology

[0002] S-palmitoylation refers to the covalent binding of long-chain fatty acids (usually 16-carbon palmitic acid) to cysteine ​​residues in proteins via thioester bonds. It is a dynamic and reversible form of protein post-translational modification that is widely present in organisms and plays an important role in regulating protein transport, localization, and stability. It is closely related to the occurrence and development of various diseases, including tumors, immune disorders, inflammation, and metabolic disorders.

[0003] Currently, experimental methods for analyzing and detecting S-palmitoylated modified proteins are mainly divided into two categories: (1) Acyl-biotinyl exchange (ABE) method; (2) Click chemistry method. The first type of acyl-biotin substitution method involves extracting cell or tissue protein samples, first blocking all free sulfhydryl groups on the cysteine ​​(Cys) residues of the protein with N-ethylmaleimide (NEM), then selectively cleaving the fatty acid (palmitic acid is the most typical) modification groups on the cysteine ​​(Cys) residues bound by thioester bonds with hydroxylamine (NH2OH). The newly generated free sulfhydryl groups react with reagents with sulfhydryl-specific reactivity (such as HPDP-biotin) to form disulfide bonds, thus labeling the newly generated free sulfhydryl groups with biotin. The biotin is then enriched and eluted using the interaction between biotin and avidin, followed by SDS-PAGE electrophoresis and high-throughput proteomic analysis to identify S-palmitoylated modified proteins (this method is mainly used to identify S-palmitoylated modified proteins). The second type of click chemistry method uses palmitic acid alkynyl alkynyl-containing palmitate analog metabolites (Alk-14) to label cultured cells (or label animal tissues). Cells or tissues are then lysed to obtain protein samples. Utilizing the high efficiency, stability, and specificity of the covalent bonds formed by the reaction of azide and alkyne, proteins with azide or alkyne groups are selectively linked to biotin or fluorescent groups. The interaction between biotin and avidin is then used for enrichment and elution. SDS-PAGE electrophoresis is then performed, followed by fluorescence signal detection or high-throughput proteomic analysis to identify S-palmitoylated modified proteins (this method is also primarily used for identifying S-palmitoylated modified proteins). The two experimental methods mentioned above mainly focus on identifying and analyzing target proteins modified by S-palmitoylation and confirming specific modification sites. As reported in Chinese patent documents CN106153941B and CN113358634A, the complex and tedious sample enrichment process, combined with Western blotting or mass spectrometry for qualitative identification, has brought significant breakthroughs to the discovery and identification of new S-palmitoylated proteins. Recent reports have found that multiple proteins, such as PD-L1, NOD1, and STAT3, are modified by S-palmitoylation and play important regulatory roles in diseases such as tumors, immune disorders, and inflammation, providing theoretical support for further research on the biological roles of these S-palmitoylated proteins in other diseases.In fact, in basic and applied research, after identifying S-palmitoylated target proteins using the two methods mentioned above (acyl-biotin substitution method and click chemistry method), further research is needed to investigate the related mechanisms and functions of S-palmitoylated target proteins. This research process involves multiple, iterative, or high-throughput quantitative analysis of S-palmitoylated target proteins, such as high-throughput screening of small molecule inhibitors targeting S-palmitoylated target proteins. Given that the two experimental methods described above for identifying S-palmitoylated proteins mainly focus on discovering and identifying new S-palmitoylated modified proteins, the experimental process is complex and cumbersome, which to some extent affects the accuracy of quantitative analysis of S-palmitoylated target proteins, and is unsuitable for high-throughput detection due to its complexity. Currently, there is no reported experimental method that can effectively perform high-throughput quantitative detection of S-palmitoylated target proteins. To meet the needs of high-throughput quantitative detection of S-palmitoylated target proteins, it is urgent to research and develop a detection kit and its usage method for high-throughput quantitative detection of S-palmitoylated target proteins. Summary of the Invention

[0004] Given that existing detection methods focus on identifying S-palmitoylated modified proteins (acyl-biotin substitution method and click chemistry method), there is currently no reported experimental method that can effectively perform high-throughput quantitative detection of S-palmitoylated modified target proteins.

[0005] Through extensive experiments and comprehensive analysis, this invention has developed a method that integrates the experimental principles of immunoprecipitation (IP), click chemistry, and enzyme-linked immunosorbent assay (ELISA) to achieve high-throughput quantitative detection of S-palmitoylated target proteins. This method is suitable for studying the mechanism of action and function of S-palmitoylated target proteins (such as high-throughput screening of small molecule inhibitors targeting S-palmitoylated target proteins), thus improving experimental efficiency.

[0006] The present invention provides an ELISA kit for quantitative detection of S-palmitoylated target proteins, comprising a magnetic device, a multi-well plate, and solutions A, B, C, D, E, F, and G.

[0007] in

[0008] Solution A is a DMSO solution of Palmitic Acid Alkyne (Alk-14);

[0009] Solution B is an NP40 lysis buffer;

[0010] Solution C is NP40 buffer solution;

[0011] Solution D is a reaction mixture;

[0012] Solution E is the signal substrate solution;

[0013] Solution F is the signal substrate responder;

[0014] Solution G is TBST buffer.

[0015] Furthermore,

[0016] Solution A is a DMSO solution of 10 mM Palmitic Acid Alkyne (Alk-14);

[0017] Solution B is a 1% NP40 lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40 (v:v), and a protease inhibitor;

[0018] Solution C is 0.1% NP40 buffer;

[0019] Solution D is a reaction mixture containing 10 mM biotin-azide, 50 mM tris(2-carbonylethyl)phosphohydrochloride, 10 mM tris(1-benzyl-1H-1,2,3-triazol-4-yl)methylamine, and 50 mM copper sulfate; (the concentrations of each component in the reaction mixture of solution D were determined through multiple experiments).

[0020] Solution E is a 1.25 mg / mL streptomycin-HRP aqueous solution, or HRP fluorescent substrate, or HRP chemiluminescent substrate;

[0021] Solution F is TMB chromogenic solution or HRP fluorescent reagent or HRP chemiluminescent reagent. Solution G is TBST buffer (10×, pH 7.4).

[0022] The magnetic device consists of Protein G magnetic beads and a porous plate magnetic structure.

[0023] The perforated plate is a 96-well plate.

[0024] Furthermore, solutions A (1 mL), B (10 mL), and C (10 mL) were stored at -20°C; solution D (3 mL) was stored at 4°C; solution E (10 μL) was stored at 4°C; and solution F (10 mL) and G (100 mL) were stored at 4°C protected from light.

[0025] The present invention also provides an experimental principle for an ELISA assay kit for quantitative detection of S-palmitoylated target proteins, including the combination of IP and Click chemistry and ELISA methods.

[0026] Furthermore, this includes the combination of Metabolic labeling and IP, Click chemistry, and ELISA experimental methods.

[0027] The method of using an ELISA kit for quantitative detection of S-palmitoylated target proteins based on the aforementioned principle includes four experimental steps: cell labeling, immunoprecipitation, click chemistry, and enzyme-linked immunosorbent assay.

[0028] The four experimental steps are as follows:

[0029] S01 labeling (Metabolic labeling) uses solution A to label the target protein overexpressed in live cells (the target protein is known to be palmitoylated).

[0030] S02 immunoprecipitation (IP) was used to lyse proteins, followed by incubation with Protein G magnetic beads and Flag antibodies to enrich the target protein.

[0031] S03 click chemistry, add TBST (1×) to enrich magnetic beads, and incubate with solution C and solution D;

[0032] S04 enzyme-linked immunosorbent assay (ELISA): Incubate with solution E, react with solution F, and after terminating the reaction, detect the absorbance, fluorescence intensity, or chemiluminescence intensity.

[0033] Furthermore, the four experimental steps are as follows:

[0034] S01 labeling (Metabolic labeling): 6-well plate cultured cells were transfected with the x gene (x refers to the gene name of the X protein with S-palmitoylation modification) eukaryotic expression plasmid. 48 hours later, solution A (diluted 1:100) was added to the cell culture medium, and the cells were cultured in an incubator for another 6 hours. This was used to label overexpressed X proteins (e.g., exogenously expressed X proteins with a Flag tag at the N-terminus).

[0035] After S02 immunoprecipitation (IP) and labeling time, add 200 μL of solution B to each well, lyse on ice for 20 minutes, transfer the lysis buffer to a 1.5 mL EP tube, centrifuge at 14000 rpm for 15 minutes at 4°C, and transfer the supernatant to a new 1.5 mL EP tube. The protein concentration of the supernatant was detected by BCA method. Then, the protein concentration of the supernatant was adjusted to ~2 μg / μL with solution B. Then, the following were added sequentially to a 96-well transparent polystyrene microplate: (1) 100 μL of protein lysis buffer sample with a concentration of 2 μg / μL, (2) Flag antibody (1:100 dilution), (3) Protein G magnetic beads (10 μL), and incubated overnight at 4°C with gentle shaking. The next day, the magnetic beads (target protein) were enriched using a 96-well plate magnetic rack, and the supernatant was discarded.

[0036] S03 Click chemistry: After washing, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, enrich magnetic beads using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times. Then add 75 μL of solution C and 25 μL of solution D to each well and incubate at room temperature for 1 hour.

[0037] For S04 enzyme-linked immunosorbent assay (ELISA), wash again, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, enrich the magnetic beads using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times; dilute solution E with 1×TBST at a ratio of (1:5000), add 100 μL to each well, and incubate at room temperature for 2 hours; wash again, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, enrich the magnetic beads using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times; then add 100 μL of solution F to each well, react at room temperature in the dark for 15-30 minutes until the expected color change occurs; stop the reaction by adding 50 μL of laboratory-prepared 2M H2SO4, and then detect the absorbance, fluorescence intensity, or chemiluminescence intensity of each well.

[0038] This invention utilizes the principle of Protein A / G binding to the Fc terminus of antibodies to magnetically adsorb and enrich S-palmitoylated target proteins onto the bottom of a 96-well plate, facilitating subsequent quantitative detection and analysis (high throughput). This invention integrates seven detection reagents (AG), employing multiple experimental principles and methods such as immunoprecipitation, click chemistry, and enzyme-linked immunosorbent assay (ELISA) to achieve high-throughput quantitative detection of S-palmitoylated target proteins. Research has validated its suitability for studying the mechanism of action and function of S-palmitoylated target proteins (such as high-throughput screening of small molecule inhibitors targeting S-palmitoylated target proteins), thus improving experimental efficiency.

[0039] The technical solution of this invention does not require complex and cumbersome centrifugation and enrichment processes, nor does it require complex and expensive mass spectrometry detection instruments. The operation process is simpler and more accurate. The entire detection process is simpler and more efficient than combined WB or mass spectrometry, with significantly lower detection costs. The sample processing method is simpler and more accurate, optimizing the quantitative detection of S-palmitoylated modified proteins and improving experimental efficiency. Attached Figure Description

[0040] Figure 1 This is a flowchart illustrating the instructions for use of an ELISA kit for the quantitative detection of S-palmitoylated target proteins.

[0041] Figure 2 The standard curve results for detecting palmitoylation modification of X protein are presented in an ELISA kit for quantitative detection of S-palmitoylated target protein.

[0042] Figure 3 This study used an ELISA kit to quantitatively analyze the S-palmitoylation modification of protein X in different treatment groups. Data are presented as mean ± standard deviation (n = 6), Control vs. 2-BP, *P < 0.01.

[0043] Figure 4 This is a schematic diagram of the mechanism of an ELISA kit for the quantitative detection of S-palmitoylated target proteins. Detailed Implementation

[0044] The technical methods of the present invention will be thoroughly reviewed and described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the embodiments described in this invention are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1:

[0046] An ELISA kit for quantitative detection of S-palmitoylated target proteins according to the present invention comprises:

[0047] [Protein G magnetic beads (1mL)]: These are commercially available Protein G magnetic beads (such as BeyoMagnet). TM Protein G beads (from other companies) are acceptable.

[0048] [96-hole transparent polystyrene microplate (1 piece)]: This is a commercially available 96-hole transparent polystyrene microplate (such as...) 96-well transparent flat-bottomed polystyrene microplates, or any multi-well plate that can be used with relevant testing instruments.

[0049] [96-hole plate magnetic rack (one piece)]: This is a commercially available 96-hole plate magnetic rack (such as BeyoMagnet). TM Magnetic separation frame), or 96-hole plate magnetic frames sold by other companies are also acceptable.

[0050]

Solution A (1 mL)

[0051] [Solution B (10 mL)]: 1% NP40 lysis buffer, containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40 (v:v), protease inhibitor (cocktail), prepared with deionized water, stored at -20°C.

[0052] [Solution C (10 mL)]: This is a 0.1% NP40 buffer solution, prepared with 0.1% NP40 (v:v) and deionized water, and stored at -20°C.

[0053] [Solution D (3 mL)]: This is the reaction mixture containing 10 mM biotin-azide (1.6 mL), 50 mM tris(2-carbonylethyl)phosphohydrochloride (0.54 mL), 10 mM tris(1-benzyl-1H-1,2,3-triazol-4-yl)methylamine (0.32 mL), and 50 mM copper sulfate (0.54 mL). It is prepared with deionized water and stored at 4 °C. (The concentrations of each component in the reaction mixture in Solution D were determined through multiple experiments. This invention tested the concentration of biotin-azide at a concentration gradient (1, 2.5, 5, 10, 20 mM). The results showed that 10 mM was close to saturation, and there was no difference between 10 mM and 20 mM. Therefore, 10 mM biotin-azide was used.)

[0054] [Solution E (10uL)]: This is a 1.25mg / mL streptomycin-HRP aqueous solution. It should be diluted 1:4000-20000 before use and stored at 4℃.

[0055]

Solution F (10mL)

[0056]

Solution G (100mL)

[0057] Example 2: Use of an ELISA kit for quantitative detection of S-palmitoylated target proteins

[0058] Specifically, the steps include the following:

[0059] 1. After transfecting cells (using HEK293T cells cultured in 6-well plates as an example) with the x gene expression plasmid (x refers to the name of the target gene of a known S-palmitoylated modified protein) for 48 hours, the experiment was divided into two groups: one control group and the other group with palmitoylation modification inhibitor (2-BP, 50 μM). Then, solution A (diluted at a ratio of 1:100) was added to the cell culture medium, and the cells were cultured in an incubator for another 6 hours to label the overexpressed target protein X (X protein is known to have S-palmitoylation modification, and the exogenously expressed X protein has a Flag tag at its N-terminus).

[0060] 2. Add 2 mL of 1×PBS phosphate buffer (pH 7.4, laboratory prepared) to each well and wash 3 times, 1 minute each time.

[0061] 3. Add 200 μL of solution B to each well, lyse on ice for 20 minutes, transfer the lysate to a 1.5 mL EP tube, centrifuge at 14000 rpm for 15 minutes at 4 °C, and transfer the supernatant to a new 1.5 mL EP tube.

[0062] 4. The protein concentration of the supernatant was detected by the BCA method. Then, the protein concentration of the supernatant was adjusted to 2 μg / μL with solution B. Then, 100 μL of protein lysis buffer sample with a concentration of 2 μg / μL was added sequentially to a 96-well transparent polystyrene microplate.

[0063] (2) Flag antibody (1:100 dilution), (3) Protein G magnetic beads (10 μL), incubated overnight at 4°C with gentle shaking, then enriched the target protein using a 96-well plate magnetic rack, and the supernatant was discarded.

[0064] 5. Discard the supernatant, wash, add 200 μL TBST (1×) to each well, incubate for 10 minutes, enrich magnetic beads (target protein) using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times.

[0065] 6. Add 75 μL of solution C and 25 μL of solution D to each well and incubate at room temperature for 1 hour.

[0066] 7. Wash, add 200 μL TBST (1×) to each well, incubate for 10 minutes, enrich magnetic beads (target protein) using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times.

[0067] 8. Dilute solution E with 1×TBST at a ratio of 1:5000, add 100 μL to each well, and incubate at room temperature for 2 hours. (A negative control well is included in this step; add the same volume of 1×TBST.)

[0068] 9. Wash, add 200 μL TBST (1×) to each well, incubate for 10 minutes, enrich magnetic beads (target protein) using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times.

[0069] 10. Add 100 μL of solution F to each well and react at room temperature in the dark for 15-30 minutes until the color changes as expected.

[0070] 11. Add 50 μL of 2M H2SO4 (laboratory preparation) to terminate the reaction, and then measure the absorbance of each well at 450 nm using a microplate reader.

[0071] Example 3: Linear Range

[0072] like Figure 2 As shown, this experiment uses the Control group sample as an example. The Control histone sample was diluted at different ratios to obtain Control histone samples of different concentrations. The obtained standard curve results show that it is linear in the range of 0-2.0 μg / μL.

[0073] Example 4: Detection of specificity:

[0074] Comparative analysis of the Control group and the 2-BP treatment group showed that the palmitoylation inhibitor (2-BP) significantly inhibited palmitoylation modification of the X protein. (See results for reference.) Figure 3 As shown, this demonstrates good specificity and its ability to accurately quantify the concentration of S-palmitoylated modified proteins.

[0075] Example 5: Technical Solution Principles and Methods (e.g.) Figure 1 and 4 (As shown)

[0076] 1. Metabolic labeling

[0077] Forty-eight hours after transfecting the x gene expression plasmid (x refers to the name of the target gene of a known S-palmitoylated modified protein) into cells (using HEK293T cells cultured in 6-well plates as an example), the cells were treated according to experimental requirements. Then, solution A (diluted at a ratio of 1:100) was added to the cell culture medium, and the cells were cultured in an incubator for another 6 hours to label the overexpressed X protein (X protein is known to have S-palmitoylation modification, and the exogenously expressed X protein has a Flag tag at its N-terminus).

[0078] 2. Immunoprecipitation (IP)

[0079] After the labeling time was completed, 200 μL of solution B was added to each well, and the mixture was lysed on ice for 20 minutes. The lysate was then transferred to a 1.5 mL EP tube and centrifuged at 14000 rpm for 15 minutes at 4 °C. The supernatant was then transferred to a new 1.5 mL EP tube. The protein concentration in the supernatant was detected by the BCA method. The protein concentration in the supernatant was then adjusted to ~2 μg / μL with solution B. Then, the following were added sequentially to a 96-well transparent polystyrene microplate: (1) 100 μL of protein lysate sample with a concentration of 2 μg / μL, (2) Flag antibody (1:100 dilution), and (3) Protein G magnetic beads (10 μL). The plate was incubated overnight at 4 °C. The magnetic beads (target protein) were then enriched using a 96-well plate magnetic rack, and the supernatant was discarded.

[0080] 3. Click Chemistry

[0081] Wash the sample, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, enrich the target protein using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times. Add 75 μL of solution C and 25 μL of solution D to each well, and incubate at room temperature for 1 hour.

[0082] 4. Enzyme-linked immunosorbent assay (ELISA)

[0083] After the previous step, wash again, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, enrich magnetic beads (target protein) using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times. Dilute solution E with 1×TBST at a ratio of 1:5000, add 100 μL to each well, and incubate at room temperature for 2 hours. Wash, add 200 μL of TBST (1×) to each well, incubate for 10 minutes, and repeat 3 times. Then, add 100 μL of solution F to each well, react at room temperature in the dark for 15-30 minutes, until the expected color change occurs. Stop the reaction by adding 50 μL of 2M H2SO4 (laboratory prepared), and then measure the absorbance of each well at 450 nm using a microplate reader.

[0084] Example 6: Improved Research

[0085] Given that solution E can be detected using streptomycin-labeled HRP corresponding absorbance values, it can also react with HRP fluorescent substrates (such as dihydrofluorescein, dihydrorhodamine, Amplite Red, etc.) or HRP chemiluminescent substrates (such as ECL substrates) to generate signals. Therefore, solution F can also be replaced by other HRP fluorescent reagents or HRP chemiluminescent reagents, and the signals can be analyzed using instruments suitable for fluorescence detection or luminescence detection, respectively.

[0086] Technical Effects: This invention utilizes the principle of Protein A / G binding to the Fc terminus of antibodies to enrich S-palmitoylated modified proteins through magnetic adsorption, facilitating subsequent quantitative detection and analysis (high throughput). This invention integrates seven detection reagents (AG) and applies multiple experimental methods, including immunoprecipitation, click chemistry, and enzyme-linked immunosorbent assay (ELISA), to achieve high-throughput quantitative detection of S-palmitoylated modified target proteins. Research shows that this invention is suitable for studying the mechanism of action and function of S-palmitoylated modified target proteins (such as high-throughput screening of small molecule inhibitors targeting S-palmitoylated modified target proteins), thus improving experimental efficiency.

[0087] The technical solution of this invention does not require complex and cumbersome centrifugation and enrichment processes, nor does it require complex and expensive mass spectrometry detection instruments. The operation process is simpler and more accurate. The entire detection process is simpler and more efficient than combined WB or mass spectrometry, with significantly lower detection costs. The sample processing method is simpler and more accurate, optimizing the quantitative detection of S-palmitoylated modified proteins and improving experimental efficiency.

[0088] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Obviously, other related modifications can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. An ELISA kit for quantitative detection of S-palmitoylated target proteins, characterized in that: The experimental principle of the ELISA kit for quantitative detection of S-palmitoylated target proteins includes the combination of metabolic labeling, IP, click chemistry, and ELISA methods. The kit includes a magnetic device, a multi-well plate, and solutions A, B, C, D, E, F, and G. Solution A (1 mL), Solution B (10 mL), and Solution C (10 mL) should be stored at -20°C; Solution D (3 mL) should be stored at 4°C; Solution E (10 µL) should be stored at 4°C; Solution F (10 mL) and Solution G (100 mL) should be stored at 4°C protected from light. The magnetic device consists of Protein G magnetic beads and a multi-well plate magnetic frame. The multi-well plate is a 96-well plate. The magnetic frame is adapted to the 96-well plate for the directional enrichment of magnetic beads during the IP process and for the absence of centrifugation washing of magnetic beads after the click chemistry reaction, so as to realize the continuous operation of the IP-click chemistry-ELISA steps in the same microplate. Solution A is a DMSO solution of 10 mM Palmitic Acid Alkyne; Solution B is a 1% NP40 lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40 (v:v), and a protease inhibitor; Solution C is 0.1% NP40 buffer; Solution D is a reaction mixture containing 10 mM biotin-azide, 50 mM tris(2-carbonylethyl)phosphohydrochloride, 10 mM tris(1-benzyl-1H-1,2,3-triazol-4-yl)methylamine, and 50 mM copper sulfate; Solution E is a 1.25 mg / mL streptomycin-HRP aqueous solution, or HRP fluorescent substrate, or HRP chemiluminescent substrate; Solution F is TMB colorimetric solution, HRP fluorescent reagent, or HRP chemiluminescent reagent; Solution G is a 10× TBST buffer solution with a pH of 7.

4. The method of using the ELISA kit for quantitative detection of S-palmitoylated target proteins includes four experimental steps: cell labeling, immunoprecipitation, click chemistry, and enzyme-linked immunosorbent assay (ELISA). Cell labeling, immunoprecipitation, click chemistry, and ELISA are respectively referred to as Metabolic labeling, IP, Click chemistry, and ELISA. The experimental steps are as follows: S01. Metabolic labeling: Using solution A to label a target protein overexpressed in live cells, wherein the target protein is palmitoylated; S02. IP: Lyse the protein, add Protein G magnetic beads and target protein antibody to incubate and enrich the target protein; S03. Click chemistry: Add 1× TBST enrichment magnetic beads, and incubate with solution C and solution D; S04. ELISA: Incubate with solution E, react with solution F, and after terminating the reaction, detect the absorbance, fluorescence intensity, or chemiluminescence intensity.

2. The ELISA kit for quantitative detection of S-palmitoylated target proteins according to claim 1, characterized in that: S01. Metabolic labeling: 48 hours after transfecting the x gene eukaryotic expression plasmid into cells cultured in 6-well plates, solution A was added to the cell culture medium at a ratio of 1:100, and the cells were cultured in an incubator for another 6 hours to label the overexpressed X protein; where x refers to the gene name of the X protein with S-palmitoylation modification, and the exogenously expressed X protein has a Flag tag at its N-terminus; S02. IP: After the labeling time is over, add 200 µL of solution B to each well, lyse on ice for 20 minutes, transfer the lysate to a 1.5 mL EP tube, centrifuge at 14000 rpm for 15 minutes at 4℃, and transfer the supernatant to a new 1.5 mL EP tube; use the BCA method to detect the protein concentration of the supernatant, and then use solution B to adjust the protein concentration of the supernatant to ~2 µg / µL. Then add the following to a 96-well transparent polystyrene microplate in sequence: (1) 100 µL of protein lysate sample with a concentration of 2 µg / µL, (2) Flag antibody diluted 1:100 with 1× TBST, (3) 10 µL of Protein G magnetic beads, and incubate overnight at 4℃ with slow shaking; the next day, enrich the magnetic beads with a 96-well plate magnetic rack, discard the supernatant, and obtain the target protein; S03. Click chemistry: Wash, add 200 µL of 1× TBST to each well, let stand for 10 minutes, enrich magnetic beads with a magnetic rack in a 96-well plate, discard the supernatant, repeat 3 times; then add 75 µL of solution C and 25 µL of solution D to each well, and incubate at room temperature for 1 hour; S04. ELISA: Wash again, add 200 µL of 1× TBST to each well, incubate for 10 minutes, enrich the magnetic beads using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times; dilute solution E with 1× TBST at a ratio of 1:5000, add 100 µL to each well, and incubate at room temperature for 2 hours. After cleaning, add 200 µL of 1× TBST to each well, let stand for 10 minutes, enrich the magnetic beads using a magnetic rack in a 96-well plate, discard the supernatant, and repeat 3 times; then, add 100 µL of solution F to each well, react at room temperature in the dark for 15-30 minutes until the expected color change occurs; stop the reaction by adding 50 µL of laboratory-prepared 2M H2SO4, and then detect the absorbance, fluorescence intensity, or chemiluminescence intensity of each well.

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