Method for detecting activity of histone methyltransferase G9a / EHMT2
By measuring the amount of H3K9me1 and H3K9me2 to detect the activity of G9a, the problem of lack of effective detection methods in the prior art is solved, efficient and accurate quantitative detection is achieved, the process is simplified and cost and time is reduced.
Patent Information
- Application Number
- CN202510167882.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art lacks effective methods to detect the activity of histone methyltransferase G9a/EHMT2, and the detection process is cumbersome and difficult to quantify.
The activity of G9a was detected by measuring the amount of products H3K9me1 and H3K9me2. The reaction system included G9a, H3 histone, active methyl provider S-adenosylmethionine and buffer was used, and semi-quantitative detection was performed using mass spectrometry and western blot experiments combined with Image J Win64 software.
Efficient and accurate quantitative detection of G9a activity is achieved, the detection process is simplified, the cost and time are reduced, and the detection accuracy is improved.
Smart Images
Figure CN119876336A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to an enzyme activity detection method, and in particular to a method for detecting the activity of histone methyltransferase G9a / EHMT2. Background Art
[0002] G9a is a lysine methyltransferase, also known as euchromatic histone-lysine methyltransferase 2 (EHMT2), which catalyzes the monomethylation and dimethylation (H3K9mel, H3K9me2) of lysine residue 9 of H3 histone. In the process of studying the relationship between G9a and renal fibrosis, the members of the research team of the present invention found that there is no mature detection method available for G9a enzyme activity, the detection process is very cumbersome, and the final activity is difficult to quantify.
[0003] The current detection methods have the following shortcomings:
[0004] ① Currently, there is no commercial G9a enzyme activity detection kit on the market, and there is no systematic method to refer to in the literature.
[0005] ② Traditional enzymatic reaction is the main principle for preparing most enzyme activity finished kits on the market. However, in the process of G9a enzyme activity detection, it is not clear what indicators should be detected to quantify or semi-quantitate G9a enzyme activity.
[0006] ③ The price of protein purification by outsourcing companies is expensive, about 6,000 yuan per sample. There are only a few references to the in vitro reaction process after purification, but there is no unified regulation on the concentration of each reagent, which requires the experimenter to explore the experimental conditions on his own and easily leads to experimental failure.
[0007] ④ Although the use of mass spectrometry to detect the products after in vitro reactions can explain the problem, it is expensive, about 3,000 yuan /
[0008] Samples, and the construction period is longer.
[0009] Therefore, there is an urgent need in the market for a method to effectively detect the activity of histone methyltransferase G9a / EHMT2. Summary of the invention
[0010] Purpose of the invention: The purpose of the present invention is to provide a method for the efficient and accurate quantitative detection of the activity of histone methyltransferase G9a / EHMT2. By improving the detection method of G9a enzyme activity, the detection process is more convenient and the detection results are easier to quantify.
[0011] Technical solution: The method for detecting the activity of histone methyltransferase G9a / EHMT2 described in the present invention detects the activity of G9a between different samples by measuring the amount of product H3K9me1 and H3K9me2.
[0012] Furthermore, the reaction system includes: G9a, H3 histone, active methyl donor S-adenosylmethionine and buffer.
[0013] Furthermore, at the beginning of the reaction, the amount of G9a is equal, the amount of the reaction substrate H3 histone is equal, the concentration of the active methyl donor S-adenosylmethionine is equal, the reaction time is equal, and the reaction buffer formula is the same.
[0014] Furthermore, methods for determining the amount of product H3K9me1 and H3K9me2 include mass spectrometry, directly detecting the amount of G9a, H3K9me1, and H3K9me2, and comparing the H3K9mel / G9a and H3K9me2 / G9a ratios between different samples to reflect the activity of G9a.
[0015] Furthermore, the method for determining the amount of product H3K9me1 and H3K9me2 includes a protein blotting experiment, performing grayscale detection of H3K9me1, H3K9me2, and H3, respectively, correcting H3K9me1 and H3K9me2 using the grayscale of the H3 internal reference, and semi-quantitating the reaction products H3K9me1 and H3K9me2.
[0016] Furthermore, the grayscale detection of H3K9me1, H3K9me2, and H3 was performed using Image J Win64 software.
[0017] Further, the following steps are included:
[0018] (1) Lysis and preparation of cell or tissue samples;
[0019] (2) Binding of antibodies to G9a;
[0020] (3) adsorption of magnetic beads and antibody-G9a conjugates;
[0021] (4) eluting the G9a protein adsorbed on the magnetic beads;
[0022] (5) BCA determination and adjustment of G9a concentration
[0023] (6) In vitro reaction of G9a with samples;
[0024] (7) Detecting the contents of the reaction products H3K9mel and H3K9me2;
[0025] (8) Process data and obtain activity.
[0026] Furthermore, in the step (6), S-adenosylmethionine is added during the reaction so that the concentration of S-adenosylmethionine in the total reaction system reaches 20 μM and 1 μg of commercial H3 histone.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] This method can semi-quantitatively detect the activity of G9a between different samples by controlling the reaction conditions and measuring the products H3K9me1 and H3K9me2. This method integrates multiple simple small experiments such as IP, BCA, and WB to semi-quantitatively detect the enzyme activity of G9a, and significantly saves money and time (results can be obtained within 3 days) with high accuracy. When it is necessary to detect the activity of G9a / EHMT2 and there is no commercial kit developed, the above method can be used to semi-quantitatively detect the enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 is the protein standard curve. After removing the outliers in Table 1, the remaining 7 groups of data were plotted, with the ordinate being the protein concentration (in micrograms / ml) and the abscissa being the absorbance. The fitting curve is a linear curve with the formula y=1776x-129.1. Any absorbance detected by the microplate reader has a corresponding protein concentration, which can be used as a reference for calculating the sample protein concentration;
[0030] Figure 2 Western blot was used to semi-quantitatively detect H3K9 methylation expression. The G9a protein eluted by acid and the commercial H3 histone were placed in the reaction system mentioned in this article in a 37°C constant temperature reaction box for 6 hours. The protein concentration obtained by adding G9a volume = 10 ng / BCA. The volume difference was made up with double distilled water. After adding the commercial protein loading buffer, it was fully denatured at 95°C for 15 minutes and Western blot detection was performed. The test results are shown in the figure. G9a is an enzyme necessary for obtaining H3K9me1 and H3K9me2 after histone H3 methylation. When the reaction conditions and time are the same, the amount of H3K9mel and H3K9me2 reflects the strength of the enzyme activity. In this study, the activity of the modeling group (T1-T3 group) is stronger than that of the experimental group (C1-C3 group). The purified G9a protein group was the positive control group, and the IgG group was the negative control group. DETAILED DESCRIPTION
[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be further described below.
[0032] The method of this embodiment is as follows:
[0033] ① Lysis and preparation of cell or tissue samples
[0034] A. Sample lysis and preparation of suspended cells: Collect cells by centrifugation at room temperature for 5 minutes at 1000xg. Resuspend and wash cells with PBS, centrifuge again under the same conditions to collect cells, aspirate the supernatant, repeat 2 times. Add 100-200μl of cell lysis buffer containing protease inhibitors for every 500,000-1 million cells. Mix the cells by blowing with the tip of the pipette, and lyse on ice for 20 minutes. Centrifuge at 12000rpm at 4℃ for 15 minutes, take the supernatant, and proceed with the subsequent immunoprecipitation reaction.
[0035] B. Lysis and preparation of adherent cell samples: Aspirate the culture medium. Rinse twice with PBS, then aspirate the remaining liquid. Add 100-200μl of cell lysis solution containing inhibitors for every 500,000-1 million cells (equivalent to one well of a 6-well plate), and lyse on ice for 20 minutes. After sufficient lysis, collect as much lysate as possible, centrifuge at 12000rpm at 4℃ for 5 minutes, take the supernatant, and then proceed to the subsequent immunoprecipitation experiment.
[0036] C. Lysis and preparation of tissue samples: Cut the tissue into small pieces. If the tissue sample itself is very small, it is not necessary to cut it again. Add 100-200μl of protein lysis buffer containing inhibitors for every 10-20 mg of tissue. Add an appropriate amount of grinding beads and grind with a fully automatic liquid nitrogen freezing grinder until fully lysed. Centrifuge at 12000rpm at 4℃ for 5 minutes, take the supernatant, and proceed to the subsequent immunoprecipitation experiment.
[0037] ② Binding of antibodies to G9a
[0038] Add an appropriate amount of antibody to the protein sample according to the ratio in the antibody (Abcam antibody catalog number abb185050) manual. Because the subsequent in vitro reaction requires at least 10ng of enzyme, it is recommended to use a slightly higher concentration so that the volume ratio of the added antibody to the total protein sample is 1:50. This step can be incubated overnight on a 4°C shaker. If you need to save time, you can incubate at room temperature on a shaker for 3 hours. The purpose is to use IP-level G9a antibodies to specifically pull G9a proteins from the total protein to form antibody-G9a conjugates.
[0039] ③ Adsorption of magnetic beads and antibody-G9a conjugate
[0040] A. Preparation of magnetic beads. It is recommended to add 20 μl of original volume IP grade magnetic beads for every 100 μl volume of total protein sample. If the total protein sample volume is large, you can divide it into 100 μl for experiment, and the rest can be stored at -80℃. You can also prepare more magnetic beads for experiment according to the volume ratio. To ensure that more G9a protein is collected, it is recommended to use more magnetic beads and samples for this step. Pipette the magnetic beads into a new EP tube, add 1ml of fresh TBST, blow and wash for a few seconds, and place it on a magnetic stand for 1 minute. Repeat 2-3 times.
[0041] B. Co-incubation of magnetic beads with antibody-G9a conjugates. Add equal volume of sample to the prepared magnetic beads and incubate on a rotary mixer at room temperature for 3 hours.
[0042] ④ Elute the G9a protein adsorbed on the magnetic beads
[0043] Remove the sample from the rotary mixer, place it on the magnetic stand for 1 minute, and try to dry the supernatant and discard it. Prepare glycine acidic elution solution and Tris-Hc1 neutralization solution according to the recipe described above (you can also directly purchase commercial reagents). Add 100 μl of glycine acidic elution solution to every 20 μl original volume of magnetic beads, incubate on a horizontal shaker at the slowest speed for 5 minutes, and immediately add 10 μl of neutralization solution. Mix by blowing with the gun tip to stop elution. Try to control the acidic elution and stop elution within 15 minutes, otherwise the acidic elution solution will be in contact with G9a for too long, which will cause damage to the enzyme activity.
[0044] ⑤BCA determination and adjustment of G9a concentration
[0045] You need to prepare your own BCA protein quantification kit and prepare a protein quantification standard curve according to the kit instructions, see Table 1. Figure 1 , determine and calculate the G9a concentration according to the standard curve formula, see Table 2, set up 3 replicate wells for each sample. If the G9a concentration is different, it is necessary to adjust it to the same concentration with cell lysis buffer.
[0046] ⑥ In vitro reaction of G9a and samples
[0047] Commercial H3 histones must be prepared. Prepare in vitro G9a reaction buffer according to the above recipe. Take about 10ng of the eluted G9a sample for the next reaction, and the excess protein sample can be stored at -80℃ for a long time. Add S-adenosylmethionine to make the S-adenosylmethionine concentration in the total reaction system reach 20μM and 1μg of commercial H3 histones, and incubate at room temperature overnight or at 37℃ for 6 hours.
[0048] ⑦ Detect the content of H3K9me 1 and H3K9me2 in the reaction products
[0049] This method controls the equal amount of G9a at the beginning of the reaction, the equal amount of the reaction substrate H3 histone, the equal concentration of the active methyl donor S-adenosylmethionine, the equal reaction time, and the same reaction buffer formula, so the amount of the reaction products H3K9me1 and H3K9me2 is only related to the activity of G9a. The activity of G9a between different samples can be semi-quantitatively detected by measuring the products H3K9me1 and H3K9me2.
[0050] A. EthD mass spectrometry analysis. Add TFA to a final concentration of 0.012% to quench the enzyme-substrate reaction and load the sample as soon as possible. This method is sensitive and can be used instead if the band is too faint after Western blotting.
[0051] B. Protein blot analysis. Add instant protein loading buffer to the reacted sample (including reaction buffer), incubate at 95°C for 15 minutes to fully denature the sample, and cool. Prepare 12.5% gel according to the instructions of the acrylamide gel preparation kit or use 12.5% precast gel. Add protein mass indicator standards and samples to the loading wells in sequence, and perform electrophoresis at 80V for 30 minutes and 100V for 1 minute.
[0052] Activate the PVDF membrane with methanol for 1 minute. Transfer the protein to the PVDF membrane with a fast transfer solution at 400 mA for 30 minutes. Block the membrane with 5% milk at room temperature and shake it at a low speed for 1 hour.
[0053] The primary antibody for H3K9me2 was incubated at 4°C overnight. After washing the PVDF membrane with TBST, the secondary antibody corresponding to the primary antibody was incubated at room temperature for 1 hour. After washing the membrane with TBST again, the membrane was placed in ECL luminescent solution for 10 seconds and imaged in the Tanon imaging system. After stripping the PVDF membrane with membrane regeneration solution, the internal reference H3 primary antibody was incubated at 4°C
[0054] After washing the PVDF membrane with TBST, incubate with the secondary antibody of the same species as the primary antibody for 1 hour at room temperature.
[0055] After washing the membrane with TBST again, the membrane was placed in ECL luminescent solution for 10 seconds and imaged in a developer.
[0056] ⑧Processing data
[0057] Mass spectrometry can directly detect the amounts of G9a, H3K9mel, and H3K9me2, and compare the H3K9mel / G9a and H3K9me2 / G9a ratios between different samples to reflect the activity of G9a.
[0058] In the protein blotting experiment, the grayscale of H3K9me1, H3K9me2, and H3 was detected by Image J Win64 software, and the grayscale of H3 internal reference was used to calibrate H3K9me1 and H3K9me2, and the reaction products H3K9me1 and H3K9me2 were semi-quantified.
[0059] The protein standard is (Yamei's Omni-Easy TM Ready-to-use BCA protein quantification kit, catalog number ZJ102).
[0060] Table 1 Preparation of standard curve
[0061] Note: After removing the outliers in Table 1, the remaining 7 groups of data were plotted, with the ordinate being the protein concentration (in micrograms / ml) and the abscissa being the absorbance. The fitting curve is a linear curve, with the formula y=1776x-129.1. Any absorbance detected by the microplate reader has a corresponding protein concentration, which can be used as a reference for calculating the sample protein concentration.
[0062] Table 2 G9a protein concentration of samples
[0063]
[0064] Note: In the IgG group, rabbit immunoglobulin IgG was added to the IP purified protein without adding the primary antibody G9a. Because the affinity of IgG to G9a is significantly weaker than that of the primary antibody to G9a, the protein concentration obtained after elution by the acid elution method should be significantly lower than that of other components. The large DDDDDDF-β1 powder was diluted to 10 ng / ml with PBS and added to the cell culture dish, and the same volume of PBS was added to the control group. Three biological replicates were set up, and the experimental groups were marked as C1, C2, and C3, and the control groups were marked as T1, T2, and T3. Therefore, the C1, C2, and C3 groups are the G9a proteins eluted from the control group before modeling, and the T1, T2, and T3 groups are the G9a proteins eluted from the experimental group after modeling. Three replicate wells were set up respectively, and the average absorbance was substituted into the formula to calculate the concentration. Because the cell amount during IP is roughly similar, the amount of lysate and primary antibody added is the same, and the overall protein concentration should be similar. After measurement, the absorbance and concentration are indeed similar (as shown in the table). The G9a group is the protein concentration of commercial recombinant human G9a protein diluted with PBS according to the instructions. The yellow-marked values are obvious outliers and should be removed in the calculation. The average absorbance is rounded to 3 decimal places.
[0065] Table 3 Grayscale measurement data of Image J software
[0066]
[0067]
[0068] Note: Image J Win 64 software was used to measure the grayscale values of the images obtained by Western blot, and all values were taken to 3 decimal places. H3 is the histone internal reference, and the ratio of H3K9me1 and H3K9me2 to the H3 internal reference is expressed as the relative expression of H3K9me2 and H3K9me2. Finally, the expression of the IgG group, i.e. the negative control group, was artificially set to 1, and the remaining calibrated expression levels are shown in columns 5 and 8 of the table. The purpose is to indirectly reflect the activity of G9a by the expression of H3K9me1 and H3K9me2.
[0069] Table 4 Source and concentration of antibodies used in Western blot
[0070]
[0071] Note: For reference when reproducing experimental results
[0072] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any technician in the relevant technical field, without departing from the scope of the technical solution of the present invention, makes any form of equivalent replacement or modification to the technical solution and technical content disclosed in the present invention, which does not depart from the content of the technical solution of the present invention and still falls within the protection scope of the present invention.
Claims
1. A method for detecting the activity of histone methyltransferase G9a / EHMT2, characterized in that: The activity of G9a in different samples was detected by measuring the amount of product H3K9me1 and H3K9me2.
2. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 1, characterized in that: The reaction system includes: G9a, H3 histone, active methyl donor S-adenosylmethionine and buffer.
3. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 2, characterized in that: At the beginning of the reaction, the amount of G9a was equal, the amount of the reaction substrate H3 histone was equal, the concentration of the active methyl donor S-adenosylmethionine was equal, the reaction time was equal, and the reaction buffer formula was the same.
4. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 1, characterized in that: Methods for measuring the amount of product H3K9me1 and H3K9me2 include mass spectrometry, directly detecting the amount of G9a, H3K9me1, and H3K9me2, and comparing the H3K9me1 / G9a and H3K9me2 / G9a ratios between different samples to reflect the activity of G9a.
5. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 1, characterized in that: The method for determining the amount of product H3K9me1 and H3K9me2 includes a protein blotting experiment, performing grayscale detection of H3K9me1, H3K9me2, and H3, respectively, correcting H3K9me1 and H3K9me2 with the grayscale of the H3 internal reference, and semi-quantitating the reaction products H3K9me1 and H3K9me2.
6. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 5, characterized in that: The grayscale detection of H3K9me 1, H3K9me2, and H3 was performed using Image J Win64 software.
7. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 5, characterized in that: The steps include: (1) Lysis and preparation of cell or tissue samples; (2) Binding of antibodies to G9a; (3) adsorption of magnetic beads and antibody-G9a conjugates; (4) eluting the G9a protein adsorbed on the magnetic beads; (5) BCA determination and adjustment of G9a concentration (6) In vitro reaction of G9a with samples; (7) Detecting the contents of H3K9me1 and H3K9me2 of the reaction products; (8) Process data and obtain activity.
8. The method for detecting the activity of histone methyltransferase G9a / EHMT2 according to claim 7, characterized in that: In the step (6), S-adenosylmethionine is added during the reaction so that the concentration of S-adenosylmethionine in the total reaction system reaches 20 μM and 1 μg of commercial H3 histone.