A high-throughput plate detection kit for detecting the palmitoylation level of a protein and a method for using the same

Abstract

The present invention provides a high-throughput well plate detection kit for detecting the palmitoylation level of a protein, the kit comprising a NEM reagent, a HA solution, a Biotin-HPDP reagent, and an ELISA plate. The present invention also provides a method for preparing the ELISA plate used in the above-mentioned kit and a high-throughput quantitative detection method for palmitoylated proteins based on the above-mentioned kit. The high-throughput well plate detection kit for detecting the palmitoylation level of a protein provided by the present invention saves about 2 days in one detection compared to the traditional method, and can detect a large number of samples at one time, and can also achieve the purpose of relative or absolute quantification, which is conducive to detecting palmitoylated proteins related to diseases and developing into a kit for clinical detection.

Description

Technical Field

[0001] The present invention relates to the field of kits, and in particular to a high-throughput well plate detection kit for detecting the palmitoylation level of a protein and a use method thereof. Background technology:

[0002] Palmitoylation of proteins is a post-translational modification process. It is a common protein modification in organisms. It mainly refers to the connection of long-chain fatty acids (usually 16-carbon palmitic acid) to cysteine ​​residues of proteins through reversible thioester bonds. This acylation modification plays a key regulatory role by affecting protein stability, subcellular localization, enzyme activity, transcriptional activity, protein-protein interaction, and protein-DNA interaction. Palmitoylation is a reversible process that enables proteins to shuttle back and forth between biological membranes and substrates in the cytoplasm, improving the efficiency of biological reactions. Studies have found that abnormal palmitoylation of proteins is widely involved in physiological processes such as metabolism, cancer, transcription, immune response, neurobiology, and signal transduction, and thus exists in the pathological mechanisms of various diseases. Recent studies have found that palmitoylated proteins are also closely related to the occurrence and development of various neurodegenerative diseases.

[0003] At present, the methods for detecting protein palmitoylation include radioactive palmitate metabolic labeling, click chemistry (metabolic labeling with azido or alkynyl palmitate analogs), mass spectrometry, acyl-biotinyl exchange (ABE) and acyl-biotinyl exchange-proximity ligation assay (ABE-PLA).

[0004] Different methods have their own characteristics: Radioactive palmitate metabolic labeling is an earlier developed method. This method has many defects: cumbersome operation and long exposure time; high concentrations of radioactive isotopes are harmful and expensive to handle; it can only detect proteins that undergo palmitoyl metabolism in living cells, and cannot detect palmitoylated proteins with low abundance; there is a lack of methods for directly enriching and identifying radiolabeled proteins. Click chemistry cannot analyze tissue samples and body fluid samples, and the introduction of palmitic acid analogs may have potential effects on cell biology. Mass spectrometry has high requirements for laboratory equipment, requiring expensive mass spectrometers and highly purified samples. ABE is a common method for detecting post-translational modifications of proteins. The sulfhydryl group on the cysteine ​​residue of a protein is highly reactive and easily undergoes various redox reactions or acylation reactions. The ABE method developed based on this is essentially to convert the post-translational modification to be detected into an acylated biotin group. ABE does not have the disadvantages of the above methods and is widely used, but the method is cumbersome to operate, the overall experimental cycle is long and the error tolerance is low. At the same time, the presence of biotin-binding proteins in the body itself may cause a slightly higher background. ABE-PLA combines PLA technology with ABE, and can be applied to the cell and tissue levels to perform spatial imaging of palmitoylation modification of proteins, reducing the false positive problem that ABE itself may have, but its high technical cost has not yet been widely used. Based on this, palmitoylation detection still generally has problems such as cumbersome operation and difficulty in quantification, so a convenient and easy-to-use kit is needed to reduce time and labor costs and improve efficiency.

[0005] At present, the Chinese patent document with the publication number CN113358634A has disclosed a detection kit for palmitoylated modified proteins and its use method. The method introduces the use of centrifuge columns, which can well reduce sample loss and shorten sample processing time for different sample processing requirements. However, the invention application does not disclose the specific properties of the centrifuge columns, and the molecular size of the trapped substances is not provided, which may not achieve the expected effect. Based on the above problems, protein palmitoylation detection is in urgent need of a sensitive, efficient and quantitative detection method.

[0006] In view of this, this invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a high-throughput plate detection kit for detecting the palmitoylation level of a protein, which can be applied to quantitatively detect the palmitoylation of proteins in tissue and body fluid samples. The kit can be used to detect palmitoylated proteins in tissues and body fluids. The kit and its application can solve the problems that conventional ABE-WB detection of protein palmitoylation is not sensitive enough, the number of single detections is small, and it is not easy to quantify. The kit has the advantages of being convenient, efficient, and quantitative.

[0008] In order to achieve the above object, the present invention provides a high-throughput well plate detection kit for detecting the palmitoylation level of a protein, characterized in that the kit comprises a NEM reagent, a HA solution, a Biotin-HPDP reagent, and an ELISA plate.

[0009] Preferably or optionally, the ELISA plate comprises a 96-well ELISA plate coated with avidin and a 96-well ELISA plate for total protein, and the 96-well ELISA plate coated with avidin is coated with avidin and blocked with 10% BSA.

[0010] Preferably or optionally, the kit further comprises a buffer, which comprises a lysis buffer, a dilution buffer, a 4SB buffer, a 2SB buffer, a -HA buffer solution, a +HA buffer solution and an HPDP reaction buffer solution. Preferably, the buffer comprises a 10× lysis buffer, a 10× dilution buffer, a 10×4SB buffer, a 10×2SB buffer, a 10×-HA buffer solution, a 10×+HA buffer solution and a 10×HPDP reaction buffer solution.

[0011] Preferably or alternatively, the 10× lysis buffer comprises 1.5 M sodium chloride, 0.5 M Tris and 50 mM EDTA, and has a pH of 7.4.

[0012] Preferably or alternatively, the 10× dilution buffer comprises 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA and 2% Triton X-100, and has a pH of 7.4.

[0013] Preferably or alternatively, the 10×4SB buffer comprises 4% SDS, 50 mM Tris and 50 mM EDTA, with a pH of 7.4; the 10×2SB buffer comprises 2% SDS, 50 mM Tris and 50 mM EDTA, with a pH of 7.4.

[0014] Preferably or alternatively, the 10×+HA buffer solution comprises 0.5 M Tris, 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0015] Preferably or alternatively, the 10×-HA buffer solution comprises 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0016] Preferably or optionally, the 10×HPDP reaction buffer solution comprises 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA, 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0017] On the other hand, the present invention also provides a method for using the above-mentioned kit, which is carried out according to the following method:

[0018] (1) diluting each buffer and preparing the required solutions;

[0019] (2) Take the tissue to be tested, add a lysis buffer reagent containing 10 mmol NEM, ultrasonically disrupt and lyse, add TritonX-100 to a final concentration of 0.2%, incubate on a shaker at 4°C for 1 hour, then centrifuge at 500g for 2 minutes, take the supernatant and determine the protein concentration therein;

[0020] (3) According to the protein concentration determined in step (2), take the supernatant containing 2 mg of protein, add cold acetone to precipitate, dissolve it with 4SB buffer containing 10 mM NEM, and then dilute it with dilution buffer containing 5 mM NEM, and shake it overnight at 4°C; (4) The product of step (3) was precipitated three times with cold acetone, and dissolved with 4SB buffer after each precipitation. For the first two times, 3 volumes of dilution buffer were added and diluted. After the third time, the sample was divided into two equal parts, one of which was added with 4 volumes of +HA buffer, and the other was added with an equal amount of -HA buffer, and mixed at room temperature for 1 hour;

[0021] (5) Precipitate the sample obtained in step (4) with cold acetone, then dissolve it with 4SB buffer, add 4 times the volume of HPDP reaction solution, and mix at room temperature for 1 h;

[0022] (6) The sample obtained in step (5) was precipitated with cold acetone, washed three times, and then dissolved in 2SB buffer. The sample was then diluted 20 times with dilution buffer, mixed by inversion at room temperature for 0.5 h, and then centrifuged at 12000 g for 3 min to obtain the supernatant. (7) 100, 50, 25, and 12.5 μL of the supernatant obtained in step (6) were taken from each sample, respectively, and added to a 96-well ELISA plate coated with avidin. The plate was incubated at 31° C. for 1 h on a shaker at 50 rpm. After completion, the sample was skimmed off and the 96-well ELISA plate was washed with washing solution for 5 min each time, for a total of four washes.

[0023] (8) Add 100 μL of the corresponding primary antibody to the well plate to which the sample was added in step (7), incubate on a shaker at 50 rpm at 31° C. for 1 h, remove the primary antibody and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times;

[0024] (9) Add 100 μL of the corresponding HRP-labeled secondary antibody to the well plate to which the sample was added in step (8), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the secondary antibody and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times;

[0025] (10) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (9), incubate at 31° C. for 5-30 min, add stop solution to each well, measure the absorbance of each well at 450 nm, record it as pA, and obtain pA(HA+) and pA(HA-);

[0026] (11) Take 100, 50, 25, and 12.5 μL of the supernatant prepared in step (6) for each sample, add it to a 96-well total protein ELISA plate, and make up each well to 100 μL with coating buffer. Let it stand overnight at 4°C. After completion, skim off the sample and wash the 96-well total protein ELISA plate with washing solution, each washing for 5 minutes, for a total of five washes;

[0027] (12) Add 300 μL of blocking solution to the well plate to which the sample was added in step (11), incubate on a shaker at 50 rpm at 31° C. for 1 h, and after completion, remove the blocking solution and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes;

[0028] (13) Add 100 μL of the corresponding primary antibody to the well plate to which the sample was added in step (12), incubate on a shaker at 50 rpm at 31° C. for 1 h, remove the primary antibody and wash the total protein 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times;

[0029] (14) Add 100 μL of the corresponding HRP-labeled secondary antibody to the well plate to which the sample was added in step (13), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the secondary antibody and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes;

[0030] (15) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (14), incubate at 31° C. for 5-30 min, add stop solution to each well, measure the absorbance of each well at 450 nm, record it as tA, and obtain tA(HA+) and tA(HA-);

[0031] (16) The palmitoylation degree of each sample was expressed as pA(HA+) / tA(HA+)-pA(HA-) / tA(HA-).

[0032] The detection flow chart of the present invention is as follows Figure 1 shown.

[0033] In the present invention, NEM refers to N-ethylmaleimide;

[0034] HA refers to hydroxylamine;

[0035] Biotin-HPDP refers to biotin HPDP-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio)propionamide, i.e. Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide;

[0036] SB refers to sodium alkyl sulfate solution, i.e. SDS buffer;

[0037] PMSF refers to phenylmethanesulfonyl fluoride, also known as phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluorid;

[0038] PI refers to protease inhibitor cocktail.

[0039] Beneficial Effects

[0040] The high-throughput well plate detection kit for detecting the palmitoylation level of a protein provided by the present invention improves the detection efficiency of protein palmitoylation and can achieve the purpose of detecting a large number of samples at one time.

[0041] This method can also further improve the accuracy of protein palmitoylation detection and achieve the purpose of quantification, saving experimental time and reducing labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is the experimental process and principle schematic diagram of the present invention;

[0043] Figure 2 This is the result before adding the stop solution in the input of the palmitoylated protein high-throughput method;

[0044] Figure 3 This is the result diagram of the palmitoylated protein high-throughput method output before adding the stop solution;

[0045] Figure 4 The results of the output and input absorbance ratios of the HA+ group and the HA- group are shown in the figure;

[0046] Figure 5 Schematic diagram of the palmitoylation detection results of APP at four dilution multiples. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0049] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0050] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0051] Example 1

[0052] This embodiment provides a high-throughput plate detection kit for detecting the palmitoylation level of a protein.

[0053] The kit comprises a buffer solution, a NEM reagent, a 50% HA solution, a Biotin-HPDP reagent, a 96-well ELISA plate, an antibody diluent and a washing solution.

[0054] The buffer includes 10× lysis buffer, 10× dilution buffer, 10× 4SB buffer, 10× 2SB buffer, 10× -HA buffer solution, 10× +HA buffer solution and 10× HPDP reaction buffer solution.

[0055] The 10× lysis buffer included 1.5 M sodium chloride, 0.5 M Tris and 50 mM EDTA, and had a pH of 7.4.

[0056] The 10× dilution buffer includes 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA and 2% Triton X-100, and has a pH of 7.4.

[0057] The 10×4SB buffer includes 4% SDS, 50 mM Tris and 50 mM EDTA, with a pH of 7.4; the 10×2SB buffer includes 2% SDS, 50 mM Tris and 50 mM EDTA, with a pH of 7.4.

[0058] The 10×+HA buffer solution includes 0.5 M Tris, 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0059] The 10×-HA buffer solution includes 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0060] The 10×HPDP reaction buffer solution includes 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA, 2% TritonX-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

[0061] Each buffer used in the subsequent examples was diluted from 10× concentration to 1× concentration before use.

[0062] The 96-well ELISA plate comprises a total protein 96-well ELISA plate and a 96-well ELISA plate coated with avidin.

[0063] The total protein 96-well ELISA plate is a conventional total protein 96-well ELISA plate in the art. The 96-well ELISA plate coated with avidin is further blocked with 10% BSA after being coated with avidin.

[0064] The Biotin-HPDP reagent is a 50Mm Biotin-HPDP solution prepared using DMSO.

[0065] The antibody diluent was a 10% BSA solution prepared using PBST.

[0066] Example 2

[0067] This example provides a method for determining the palmitoylation level of a protein using the kit provided in Example 1.

[0068] Before the assay, the lysis buffer containing 10 mmol NEM, the 4SB buffer containing 10 mM NEM, and the dilution buffer containing 5 mM NEM were prepared using the relevant reagents in the kit.

[0069] (1) About 0.7 g of hemicerebral cortical tissue was taken from WT mice (control group, referred to as WT) and ZDHHC21 mutant mice constructed by CRISPER Cas9 technology in our laboratory (palmitoylation abnormal group, referred to as MUT), and 1 mL of lysis buffer containing 10 mmol NEM was added. Ultrasonic disruption was performed, and 30% TritonX-100 solution was added to make the final concentration of 1.7%. The mixture was incubated in a shaking incubator at 4°C for 1 h, and then centrifuged at 500 g for 2 min. The supernatant was taken and the protein concentration was determined.

[0070] (2) According to the protein concentration determined in step (1), take the supernatant containing 2 mg of protein, add cold acetone to precipitate, dissolve it with 300 μL of 4SB buffer containing 10 mM NEM, and then dilute it with 1200 μL of dilution buffer containing 5 mM NEM, and shake it at 4°C overnight;

[0071] (3) The product of step (2) was precipitated three times with cold acetone, and dissolved with 150 μL 4SB buffer after each precipitation. Three volumes of dilution buffer were added for dilution in the first two times. After the third time, the sample was divided into two equal parts, one part was added with 1200 μL +HA buffer, and the other part was added with 1200 μL -HA buffer, and mixed at room temperature for 1 hour;

[0072] (4) The sample obtained in step (3) was precipitated with cold acetone, then dissolved with 150 μL 4SB buffer, and 600 μL HPDP reaction solution was added and mixed at room temperature for 1 h;

[0073] (5) Using cold acetone to precipitate the sample obtained in step (4), wash the precipitate three times, add 100 μL of 2SB buffer to dissolve, then dilute 20 times with dilution buffer, mix at room temperature for 0.5 h, and then centrifuge at 12000g for 3 min to collect the supernatant;

[0074] (6) Take 100, 50, 25, and 12.5 μL of the supernatant prepared in step (5) for each sample, add it to a 96-well ELISA plate coated with avidin, and incubate it on a shaker at 50 rpm at 31°C for 1 h. After completion, skim off the sample and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times;

[0075] (7) Add 100 μL of primary antibody against mouse APP protein (Invitrogen, 14-9749-82) to the well plate to which the sample was added in step (6), and incubate at 31°C for 1 h on a shaker at 50 rpm. After completion, remove the primary antibody and wash the 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes;

[0076] (8) Add 100 μL of HRP-labeled goat anti-mouse secondary antibody to the well plate to which the sample was added in step (7), incubate on a shaker at 50 rpm at 31° C. for 1 h, remove the secondary antibody after completion, and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times;

[0077] (9) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (8), incubate at 31° C. for 5-30 min, add 50 μL of stop solution to each well, and measure the absorbance of each well at 450 nm, which is recorded as pA;

[0078] (10) Take 100, 50, 25, and 12.5 μL of the supernatant prepared in step (5) for each sample, add it to a total protein 96-well ELISA plate, and make up each well to 100 μL with coating buffer. Let it stand overnight at 4°C. After completion, skim off the sample and wash the total protein 96-well ELISA plate with washing solution, each washing for 5 minutes, for a total of five washes;

[0079] (11) Add 300 μL of blocking solution to the well plate with the sample added in step (10), incubate at 31° C. for 1 h on a shaker at 50 rpm. After completion, remove the blocking solution and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of four washes;

[0080] (12) Add 100 μL of primary antibody against mouse APP protein to the well plate to which the sample was added in step (11), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the primary antibody and wash the 96-well ELISA plate for total protein with a washing solution for 5 min each time, for a total of four washes;

[0081] (13) Add 100 μL of HRP-labeled goat anti-mouse secondary antibody to the well plate to which the sample was added in step (12), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the secondary antibody and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes;

[0082] (14) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (13), incubate at 31° C. for 5-30 min, add stop solution to each well, and measure the absorbance of each well at 450 nm, which is recorded as tA;

[0083] (15) The calculated results were obtained by subtracting the absorbance value of the blank well from the result of each sample. The degree of palmitoylation of each sample was expressed as pA(HA+) / tA(HA+)-pA(HA-) / tA(HA-) (during the reaction, the palmitoylation modification of the protein was replaced by biotinylation modification. In this formula, pA(HA+) refers to the absorbance of biotinylated APP in the +HA group; pA(HA-) refers to the absorbance of biotinylated APP in the -HA group; tA(HA+) refers to the absorbance of total APP in the +HA group; tA(HA-) refers to the absorbance of total APP in the -HA group).

[0084] The specific measurement results are shown in Table 1.

[0085] Table 1 Measurement results data table

[0086] APP-output WT-1+HA WT-2+HA WT-3+HA WT-1-HA WT-2-HA WT-3-HA (-Blank well absorbance) Sample volume 1 2 3 4 5 6 A 100μL 1.1796 1.7325 0.9417 1.3446 1.5535 0.8989 B 25μL 1.0746 1.3462 0.606 0.8666 1.1151 0.5485 C 12.5μL 0.8406 1.1606 0.5119 0.6048 0.7132 0.326 D 6.25μL 0.6587 0.9462 0.3815 0.3449 0.39 0.1945 APP-output MUT-1+HA MUT-2+HA MUT-3+HA MUT-1-HA MUT-2-HA MUT-3-HA (-Blank well absorbance) Sample volume 7 8 9 10 11 12 A 100μL 0.9845 0.8471 0.9364 0.835 0.8696 0.7534 B 25μL 0.8632 0.6754 0.8017 0.6774 0.6521 0.5614 C 12.5μL 0.7234 0.8004 0.5984 0.399 0.4344 0.3692 D 6.25μL 0.6723 0.6655 0.4741 0.2277 0.2553 0.2187 APP-input WT-1+HA WT-2+HA WT-3+HA WT-1-HA WT-2-HA WT-3-HA (-Blank well absorbance) Sample volume 1 2 3 4 5 6 A 100μL 0.3697 0.3642 0.3296 0.3929 0.363 0.3436 B 25μL 0.3542 0.3359 0.2667 0.3848 0.2514 0.314 C 12.5μL 0.2037 0.1732 0.1493 0.1382 0.1274 0.1667 D 6.25μL 0.0885 0.0858 0.0544 0.0931 0.0682 0.0652 APP-input MUT-1+HA MUT-2+HA MUT-3+HA MUT-1-HA MUT-2-HA MUT-3-HA (-Blank well absorbance) Sample volume 7 8 9 10 11 12 A 100μL 0.2731 0.2743 0.3206 0.2577 0.309 0.2692 B 25μL 0.2243 0.1981 0.287 0.2468 0.2422 0.2437 C 12.5μL 0.1209 0.1095 0.1481 0.1184 0.1377 0.1188 D 6.25μL 0.0651 0.0663 0.0758 0.0626 0.068 0.1017 output / input Sample volume WT-1+HA WT-2+HA WT-3+HA WT-1-HA WT-2-HA WT-3-HA pA / tA 100μL 3.1906 4.7570 2.8570 3.4222 4.2796 2.6161 25μL 3.0338 4.0077 2.2722 2.2520 4.4355 1.7468 12.5μL 4.1266 6.7009 3.4286 4.3762 5.5981 1.9556 6.25μL 7.4429 11.0279 7.0128 3.7046 5.7184 2.9831 output / input Sample volume MUT-1+HA MUT-2+HA MUT-3+HA MUT-1-HA MUT-2-HA MUT-3-HA pA / tA 100μL 3.6049 3.0882 2.9207 3.2402 2.8142 2.7986 25μL 3.8484 3.4093 2.7933 2.7447 2.6924 2.3036 12.5μL 5.9834 7.3095 4.0405 3.3699 3.1546 3.1077 6.25μL 10.3271 10.0377 6.2546 3.6373 3.7544 2.1504

[0087] In this embodiment, the state of the total protein 96-well ELISA plate before adding the stop solution is as follows: Figure 2 As shown in the figure, the state of the 96-well ELISA plate coated with avidin before adding the stop solution is as follows Figure 3 shown.

[0088] The results of the measurements at different concentrations are shown in Figure 5 And as shown in Table 2.

[0089] Table 2 Sample palmitoylation degree results

[0090] Sample volume WT-1+HA WT-2+HA WT-3+HA Mean 100μL -0.231549688 0.477387322 0.240976 0.162271 25μL 0.781800162 -0.42782046 0.525401 0.293127 12.5μL -0.249609432 1.102807618 1.473058 0.775419 6.25μL 3.738319164 5.309496955 4.029739 4.359185 Sample volume MUT-1+HA MUT-2+HA MUT-3+HA Mean 100μL 0.364704843 0.273985089 0.122111 0.2536 25μL 1.103684721 0.716986225 0.489728 0.770133 12.5μL 2.61352497 4.154904945 0.932769 2.567066 6.25μL 6.689808748 6.283295626 4.104175 5.692426

[0091] Depend on Figure 5 It can be seen from the results in Table 2 that the kit of the present invention can provide effective results at different concentrations and has good measurement stability and accuracy.

[0092] It can be seen from the above results that the absorbance results of palmitoylated protein and total protein measured by the kit provided in Example 1 can relatively quantitatively represent the palmitoylation degree of the protein. The above results prove that the kit and method provided by the present invention can relatively quantitatively detect the degree of palmitoylation of protein and compare the difference in the degree of palmitoylation of specific protein between different groups. Further, the kit provided by the present invention can achieve the purpose of absolutely quantitatively detecting palmitoylated APP or other proteins after further preparing a standard curve.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for using a high-throughput plate detection kit for detecting the palmitoylation level of a protein, characterized in that: The kit includes NEM reagent, HA solution, Biotin-HPDP reagent, and ELISA plate; The ELISA plate includes a 96-well ELISA plate coated with avidin and a 96-well ELISA plate for total protein, wherein the 96-well ELISA plate coated with avidin is coated with avidin and blocked with 10% BSA; Proceed as follows: (1) diluting each buffer and preparing the required solutions; (2) Take the tissue to be tested, add a lysis buffer reagent containing 10 mmol NEM, ultrasonically disrupt and lyse, add TritonX-100 to a final concentration of 0.2%, incubate on a shaker at 4°C for 1 hour, then centrifuge at 500g for 2 minutes, take the supernatant and determine the protein concentration therein; (3) According to the protein concentration determined in step (2), take the supernatant containing 2 mg of protein, add cold acetone to precipitate, dissolve it in 4SB buffer containing 10 mM NEM, and then dilute it with dilution buffer containing 5 mM NEM, and shake it at 4°C overnight; (4) The product of step (3) was precipitated three times with cold acetone, and dissolved with 4SB buffer after each precipitation. Three volumes of dilution buffer were added for dilution in the first two times. After the third time, the sample was divided into two equal parts, one of which was added with 4 volumes of +HA buffer, and the other was added with an equal amount of -HA buffer, and mixed at room temperature for 1 hour; (5) Precipitate the sample obtained in step (4) with cold acetone, then dissolve it with 4SB buffer, add 4 times the volume of HPDP reaction solution, and mix at room temperature for 1 h; (6) Precipitate the sample obtained in step (5) with cold acetone, wash the sample three times, dissolve it in 2SB buffer, dilute it 20 times with dilution buffer, mix it upside down at room temperature for 0.5 h, centrifuge it at 12000g for 3 min, and take the supernatant; (7) Take 100, 50, 25, and 12.5 μL of the supernatant prepared in step (6) for each sample, add it to a 96-well ELISA plate coated with avidin, and incubate it on a shaker at 50 rpm at 31°C for 1 h. After completion, skim off the sample and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of four times; (8) Add 100 μL of the corresponding primary antibody to the well plate to which the sample was added in step (7), incubate on a shaker at 50 rpm at 31° C. for 1 h, remove the primary antibody and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times; (9) Add 100 μL of the corresponding HRP-labeled secondary antibody to the well plate to which the sample was added in step (8), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the secondary antibody and wash the 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times; (10) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (9), incubate at 31° C. for 5-30 min, add stop solution to each well, measure the absorbance of each well at 450 nm, record it as pA, and obtain pA(HA+) and pA(HA-); (11) Take 100, 50, 25, and 12.5 μL of the supernatant prepared in step (6) for each sample, add it to a 96-well total protein ELISA plate, and make up each well to 100 μL with coating buffer. Let it stand overnight at 4°C. After completion, skim off the sample and wash the 96-well total protein ELISA plate with washing solution, each washing for 5 minutes, for a total of five washes; (12) Add 300 μL of blocking solution to the well plate to which the sample was added in step (11), incubate on a shaker at 50 rpm at 31° C. for 1 h, and after completion, remove the blocking solution and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes; (13) Add 100 μL of the corresponding primary antibody to the well plate to which the sample was added in step (12), incubate on a shaker at 50 rpm at 31° C. for 1 h, remove the primary antibody and wash the total protein 96-well ELISA plate with washing solution, washing for 5 min each time, for a total of five times; (14) Add 100 μL of the corresponding HRP-labeled secondary antibody to the well plate to which the sample was added in step (13), incubate on a shaker at 50 rpm at 31° C. for 1 h. After completion, remove the secondary antibody and wash the total protein 96-well ELISA plate with washing solution for 5 min each time, for a total of five washes; (15) Add 100 μL of TMB colorimetric solution to the well plate to which the sample was added in step (14), incubate at 31° C. for 5-30 min, add stop solution to each well, measure the absorbance of each well at 450 nm, record it as tA, and obtain tA(HA+) and tA(HA-); (16) The palmitoylation degree of each sample was expressed as pA(HA+) / tA(HA+)-pA(HA-) / tA(HA-).

2. The method of use according to claim 1, characterized in that: The kit further comprises a buffer solution, which comprises a lysis buffer, a dilution buffer, a 4SB buffer, a 2SB buffer, a -HA buffer solution, a +HA buffer solution and an HPDP reaction buffer solution.

3. The method of use according to claim 1, characterized in that: The buffer includes 10× lysis buffer, 10× dilution buffer, 10× 4SB buffer, 10× 2SB buffer, 10× -HA buffer solution, 10× +HA buffer solution and 10× HPDP reaction buffer solution.

4. The method of use according to claim 3, characterized in that: The 10× lysis buffer included 1.5 M sodium chloride, 0.5 M Tris and 50 mM EDTA, and had a pH of 7.

4.

5. The method of use according to claim 3, characterized in that: The 10× dilution buffer includes 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA and 2% Triton X-100, and has a pH of 7.

4.

6. The method of use according to claim 3, characterized in that: The 10×4SB buffer includes 4% SDS, 50mM Tris and 50mM EDTA, with a pH of 7.4; the 10×2SB buffer includes 2% SDS, 50mM Tris and 50mM EDTA, with a pH of 7.

4.

7. The method of use according to claim 3, characterized in that: The 10×+HA buffer solution includes 0.5M Tris, 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.

4.

8. The method of use according to claim 3, characterized in that: The 10×-HA buffer solution includes 2% TritonX-100, 10 mM PMSF, 10×PI, and has a pH of 7.

4.

9. The method of use according to claim 3, characterized in that: The 10×HPDP reaction buffer solution includes 1.5 M sodium chloride, 0.5 M Tris, 50 mM EDTA, 2% Triton X-100, 10 mM PMSF, 10×PI, and has a pH of 7.4.

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