Method for rapidly detecting protein content in rubber tree latex based on fluorescent probe labeling

Abstract

The invention belongs to the field of protein content detection, and discloses a rubber tree latex protein content rapid detection method based on fluorescent probe labeling, and the method comprises the following steps: selecting a latex sample, pre-treating, and diluting; a protein fluorescent dye is used for dyeing; collecting a fluorescence signal from the dyed latex sample to be detected; latex proteins with different masses are selected as standard samples for protein quantification, and fluorescence signals are collected by using the same parameters for sample detection; performing linear fitting by taking the abscissa as the latex protein mass and the ordinate as the fluorescence value to obtain a latex protein linear equation; and substituting the measured latex fluorescence signal into a latex protein linear equation, and calculating the content of the latex protein. According to the method, the protein content in the rubber tree latex is accurately and rapidly detected through the fluorescent probe labeling technology, rapid detection of the protein content in the latex is achieved, and the modern industrial production requirement for high-quality latex is met.

Description

Technical Field

[0001] This invention relates to the field of protein content detection technology, and in particular to a rapid detection method for protein content in rubber tree latex based on fluorescent probe labeling. Background Technology

[0002] Natural rubber, an important industrial raw material, is processed from the latex discharged after the latex cells of rubber trees are cut. Its main component is polyisoprene, and it also contains a small amount of non-rubber components. Protein, as a non-rubber component with a relatively high content in latex, plays a role similar to an "auxiliary agent" in natural rubber. Its content has a significant impact on improving the processing performance, mechanical properties, and aging resistance of rubber.

[0003] The current method for determining the protein content of latex adopts the Kjeldahl method specified in GB / T8088-2008 "Determination of Nitrogen Content in Natural Raw Rubber and Natural Latex". This method uses a catalyst to digest the sample with concentrated sulfuric acid, decomposing organic nitrogen into ammonia, which then combines with sulfuric acid to form ammonium bisulfate. The ammonia is then distilled off by heating with sodium hydroxide. The released ammonia is absorbed with sulfuric acid or boric acid solution, and the excess acid is titrated with the corresponding standard solution. The nitrogen content in the latex sample is calculated based on the amount consumed, and then converted to protein content. Other detection methods, such as the Folin-Ciocalteu method, biuret method, Coomassie brilliant blue method, and BCA method, are also commonly used for protein detection. These methods require sample pretreatment, are time-consuming, and involve cumbersome analytical processes. Some methods produce toxic or harmful gases during the reaction, which can easily harm human health and the environment. Furthermore, these methods require high levels of experimental skill and conditions, making them suitable only for laboratory settings and not for large-scale, rapid latex protein detection in industrial production processes.

[0004] Cellular fluorescence assays, due to their high sensitivity and specificity, as well as their advantages of accurate data, rapid detection, and cost-effectiveness, are widely used for the quantitative analysis of proteins in cells. However, the use of fluorescent dyes for the detection of proteins in rubber latex faces the following technical challenges: 1. Fresh latex is very easy to coagulate under normal temperature conditions or when improperly handled (acidic conditions), which leads to uneven sample distribution, blockage of sample dispensing equipment, and inability to perform stable fluorescence detection.

[0005] 2. Parameters for fluorescent dyes in cellular protein detection (such as dye type, concentration, and staining time) cannot be directly applied to latex systems with completely different compositions and morphologies. A new set of optimal detection conditions specifically for the latex protein-fluorescent dye reaction system needs to be established to ensure detection sensitivity, linear range, and repeatability.

[0006] 3. Challenges in preparing quantitative standards and establishing standard curves: Pure standards representing the overall characteristics of latex proteins are required extracted from the complex latex. It is essential to ensure that the preparation method of the standards is reliable, reproducible, and that their fluorescence behavior is consistent with the proteins in the latex sample being tested, in order to establish a standard curve for accurate quantification. Summary of the Invention

[0007] To address the aforementioned technical challenges, the present invention aims to provide a rapid detection method for protein content in rubber tree latex based on fluorescent probe labeling. This method accurately and rapidly detects the protein content in rubber tree latex using fluorescent probe labeling technology, thereby achieving rapid detection of latex protein content and meeting the demands of modern industrial production for high-quality latex.

[0008] To achieve the above objectives, the technical solution adopted by this invention is as follows: a rapid detection method for protein content in rubber tree latex based on fluorescent probe labeling, comprising the following steps: S1. Sample Selection and Pretreatment: Fresh latex was collected from the field and transported on ice to prevent coagulation. After filtering the latex through an 80-mesh sieve to remove impurities, the latex sample was accurately weighed into a centrifuge tube and diluted with distilled water. The latex mass was M. N The unit is mg, and the dilution factor is N.

[0009] S2. Staining of the sample: Add protein fluorescent dye to the diluted latex obtained in step S1, mix gently and let stand for more than 30 minutes to allow the dye to stain fully, with a final volume of V.

[0010] S3. Sample Detection: Aliquot 0.2 mL of the stained latex obtained in step S2 into microplates, perform fluorescence detection using a multi-functional microplate reader, and record the fluorescence value Y. L .

[0011] S4. Establishment of the standard curve: Select latex protein of different masses as standard samples for protein quantification. Collect fluorescence signals using the same parameters as the sample detection. With the x-axis representing the mass of latex protein and the y-axis representing the fluorescence value, the linear equation for the mass of latex protein is obtained as Y = aX + b.

[0012] S5. Calculation of protein mass in latex: Calculate the protein mass in latex using the following formula: M L =(Y L -b) / a, where M L Y represents the mass of latex protein, in μg. L The fluorescence value is the value of the latex to be tested.

[0013] S6. Calculation of protein content in latex: The protein content of latex is calculated according to the following formula: F = (M L×N×V) / (M N ×0.2)×100%, where F is the latex protein content, in %; M L The values ​​represent the mass of latex protein, in μg; N represents the latex dilution factor; V represents the total volume of the latex stained in step S2, in mL; and M represents the total volume of the latex. N The value represents the mass of latex, expressed in mg.

[0014] Optionally, in step S1, 1 mL (approximately 800-900 mg) of latex is accurately weighed into a centrifuge tube, and the dilution factor is between 50 and 200. The diluent is distilled water or a weakly alkaline buffer solution (to prevent latex from coagulating).

[0015] Preferably, in step S1, the latex is diluted by a factor of 100, and the diluent can be 0.1×PBS buffer.

[0016] Optionally, in step S1, the same sample is collected three times consecutively.

[0017] Optionally, in step S2, the protein fluorescent dye can be selected from cyanogenamide dye Cy5, CD3 mouse monoclonal antibody (CD3 Mouse mAb, AF488), recombinant streptavidin protein (AF594), etc., and the final volume of S2 is 1 mL.

[0018] Preferably, in step S2, Cy5 is selected as the protein fluorescent dye, the working concentration of Cy5 dye is 0.5-3.0 μM, and the staining time is 60-150 min.

[0019] More preferably, in step S2, the optimal working concentration of Cy5 is 2.0 μM and the optimal staining time is 100 min.

[0020] Optionally, in step S3, a black bottom permeable microplate is selected, the excitation light is 650nm, the emission light is 670nm, and the operating temperature of the multifunctional microplate reader is 25℃.

[0021] Optionally, in step S4, the preparation method of the latex protein standard solution includes the following steps: S41. Mix the latex with the protein extraction solvent in the specified ratio and shake to mix thoroughly.

[0022] S42. Add benzyl sulfonyl fluoride (PMSF) protease inhibitor to the mixture from step S41.

[0023] S43. Let the mixture from step S42 stand at room temperature for at least 1 hour.

[0024] S44. The mixture obtained in step S43 is centrifuged at 4°C to separate into layers.

[0025] S45. Take the intermediate clear liquid obtained in step S44 and place it in a new centrifuge tube. Centrifuge at 4°C to separate the layers.

[0026] S46. Take the intermediate supernatant of the liquid obtained in step S45 and place it in a new centrifuge tube. This is the latex protein extract. Since the protein extract contains particle detergent, the protein content is detected by BCA method.

[0027] Optionally, in step S41, the protein extraction solvent (pH=7.5) is: 7M urea, 2M thiourea, 40 mM Tris, 4% (W / V) Triton X-100, 0.07% (W / V) β-mercaptoethanol, and the volume ratio of latex to protein extraction solution is 1:3.

[0028] Preferably, in step S41, the latex should be added drop by drop and shaken in time to prevent the latex from solidifying instantly and causing surface sealing.

[0029] Optionally, the concentration of PMSF in step S42 is 100 mM, and the volume ratio of PMSF to the mixture in step S41 is 1:200.

[0030] Optionally, in step S43, the settling time is 1-2 hours.

[0031] Optionally, in step S44, the centrifuge speed is 14000-18000 rpm and the centrifugation time is 45-60 min.

[0032] Preferably, in step S44, the centrifuge speed is 18000 rpm and the centrifugation time is 60 min.

[0033] Optionally, in step S45, the centrifuge speed is 14000-18000 rpm and the centrifugation time is 15-30 min.

[0034] Preferably, in step S45, the centrifuge speed is 14000 rpm and the centrifugation time is 15 min.

[0035] Optionally, in step S5, the RSD of the three latex protein quality test results is less than 5%.

[0036] Preferably, the RSD is less than 3%.

[0037] More preferably, the RSD is less than 2%.

[0038] In summary, through systematic research and technological optimization, this invention has overcome the technical barriers mentioned in the background section one by one: 1. To address the issue of adaptability of fluorescence detection parameters, this invention has conducted comprehensive dye screening and optimization of key parameters: ① Dye Screening and Validation: By comparing the performance of three common protein fluorescent dyes, Cy5, AF488, and AF594, Cy5 was selected as the specific dye. This dye exhibits better linearity, shorter staining time, and lower cost-effectiveness in latex systems.

[0039] ② Precise optimization of key parameters: The effective range for latex concentration detection was clearly defined as 0.2%-4.0%, providing precise guidance for sample pretreatment dilution; by setting four gradients of 0.5, 1.0, 2.0, and 3.0 μM, the optimal working concentration of Cy5 was determined to be 2.0 μM. At this concentration, the widest linear detection range (latex concentration 0.2%-4.0%) and the highest correlation coefficient (R²) were obtained. 2 =0.997); Through detailed experiments (30-180 minutes, measured every 10 minutes), the optimal staining time for Cy5 was determined to be 100 minutes, at which point the fluorescence value showed an excellent correlation with protein content (R0.997). 2 >0.99), balancing the need for rapid detection and accuracy.

[0040] 2. To address the issues of solidification and impurity interference during sample processing and ensure sample stability, this invention establishes a standardized pretreatment procedure: ① Anti-coagulation treatment: Transport and store on ice, dilute with weak alkaline buffer (such as 0.1×PBS).

[0041] ②Removing impurities: Use an 80-mesh sieve for filtration.

[0042] ③ Standardized operation: Latex mass range (800-900mg), latex dilution ratio (preferably 100 times), and mixture detection volume (0.2mL) ensure the reproducibility of the operation.

[0043] 3. To establish a reliable calibration system, this invention implements the following steps: ① Preparation of standard: The latex protein standard solution was extracted and purified by differential centrifugation combined with nonionic detergent, and the purity of the standard was ensured by BCA method.

[0044] ② Establishment of standard curve: Using the self-made latex protein standards of different masses (5.53 μg to 55.3 μg) mentioned above, the fluorescence value was measured under the same detection parameters as the test sample, and a linear equation was established between the fluorescence value (Y) and the protein mass (X) (Y = 48978X + 367450, R0). 2=0.997). This curve is highly specific and directly correlates with latex proteins, rather than general proteins.

[0045] 4. Method Validation and Advantages The accuracy of this invention is verified by comparison with the BCA method (see Table 4); the invention overcomes the disadvantages of traditional methods such as complexity, time consumption, and use of toxic solvents, and achieves rapid, safe, and environmentally friendly detection, demonstrating its advantages and innovation.

[0046] In summary, this invention has developed a complete, reliable, rapid, safe, and superior latex protein detection solution compared to existing technologies through optimization of the detection system's adaptability, improvement of sample processing and stability, and accurate and reliable quantitative results. It possesses outstanding practicality and significant technological advancements.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to apply fluorescent dye technology to the detection of latex protein in natural rubber, an industrial raw material. It confirms the feasibility of this method for protein detection in the industrial raw material field. At the same time, by optimizing the detection system, the detection results of latex protein can exhibit good stability (RSD value less than 5%), ensuring rapid and accurate detection of protein content in latex.

[0048] 2. The detection method of the present invention reduces the sample pretreatment process of traditional protein detection methods, avoids the use of hazardous chemical reagents and environmental pollution, shortens the detection time, and improves the accuracy and safety of the detection results. Attached Figure Description

[0049] Figure 1 The linear relationships between three different protein fluorescent dyes and latex concentration are shown, where a: linear relationship between Cy5 fluorescent dye and latex concentration; b: linear relationship between AF488 fluorescent dye and latex concentration; c: linear relationship between AF594 fluorescent dye and latex concentration.

[0050] Figure 2 The standard curves for latex protein standard samples of different qualities are shown. Detailed Implementation

[0051] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation schemes and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0052] Example 1 Detection of protein content in rubber latex using protein fluorescent dyes 1. Test Methods 1.1 Determination of the quality of latex protein standards After filtering fresh rubber latex through an 80-mesh sieve to remove impurities, accurately pipette 0.5 mL of fresh latex and add it dropwise to 1.5 mL of protein extraction solvent (protein extraction solvent (pH=7.5) consists of: 7M urea, 2M thiourea, 40mM Tris, 4% (w / v) Triton X-100, and 0.07% (w / v) β-mercaptoethanol). Vortex to mix and let stand for 1-2 h. Centrifuge at 18000 rpm for 60 min at 4 ℃, separate the latex layer, and collect the supernatant to a new centrifuge tube. Centrifuge at 14000 rpm for 15 min at 4 ℃, and collect the supernatant to a new centrifuge tube. The resulting liquid is the protein solution from the latex. Using a BCA kit, after preheating the reaction solution to 60 ℃ for 0.5 h, add the protein solution from the latex and protein standards separately. After reacting at 60 ℃ for 0.5 h, the protein content of the latex can be measured using a microplate reader.

[0053] 1.2 Acquisition of Fluorescence Signals 1.2.1 Sample processing methods After filtering fresh rubber latex through an 80-mesh sieve to remove impurities, 1 mL of latex sample was transferred to a 2 mL centrifuge tube and weighed, denoted as M. N The unit is mg. Add an equal volume of 0.1×PBS buffer for the first dilution. Based on the required final latex concentration, pipette the first-diluted latex into a 1.5mL centrifuge tube, add the corresponding volume of 0.1×PBS buffer for the second dilution, and finally add 10μL of protein fluorescent dye to bring the reaction volume to 1mL. Incubate the reaction at room temperature in the dark for at least 60 minutes before collecting fluorescence signals. The same sample can be collected up to 3 times consecutively.

[0054] 1.2.2 Methods for Acquiring Fluorescence Signals Following the operating instructions of the SpectraMax i3x multi-functional microplate reader, first, pipette 200 μL of the reaction solution into a black-bottomed, transparent 96-well microplate, and place the microplate into the microplate reader. Select the low fluorescence intensity reading mode and the kinetic detection mode. Choose the excitation and emission wavelengths according to the fluorescent dye, and set the detection reaction time, instrument temperature (operating temperature 25℃), and reading method. Take three replicates for each sample. Fluorescence data are expressed as mean ± standard deviation.

[0055] 1.3 Optimization of Fluorescence Detection Parameters The main parameters for fluorescence detection include: dye selection, staining time, dye concentration, and latex concentration. Based on the characteristics of latex proteins, three common protein fluorescent dyes were selected: Cy5 (Cyanine5, excitation 650nm, emission 670nm), AF488 (excitation 495nm, emission 520nm), and AF594 (excitation 590nm, emission 620nm). Staining time ranged from 60 to 150 minutes, with detection every 10 minutes, for a total of 10 gradients. Fluorescent dye concentrations were set at 0.5 μM, 1.0 μM, 2.0 μM, and 3.0 μM, for a total of 4 gradients. Latex concentrations were set at 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2%, 1.6%, 2.0%, 3.0%, 4.0%, and 6.0%, for a total of 12 gradients. The linear relationship between fluorescence signal intensity and different latex concentrations was compared.

[0056] 1.4 Plotting the Standard Curve Pure latex protein solutions with different protein contents (5.53 μg, 11.06 μg, 16.59 μg, 22.12 μg, 33.18 μg, 44.24 μg, and 55.30 μg) were weighed. The linear equation Y=aX+b was established by least squares regression with protein mass as the x-axis and protein fluorescence value as the y-axis.

[0057] 1.5 Calculation of Latex Protein Mass Based on the fluorescence value Y generated when detecting samples of different latex protein quality samples. L Substitute into the following formula to calculate: M L =(Y L -b) / a Where M L The value represents the protein mass of the latex, expressed in μg.

[0058] 1.6 Calculation of Latex Protein Content The protein content of latex is calculated using the following formula: F = (M) L ×N×V) / (M N (×0.2)×100% Where F represents the latex protein content, in percentages (%), and M represents the latex protein content. L The value represents the mass of latex protein, in μg; N represents the latex dilution factor; V represents the total volume of latex stained in step S2, in mL; M N The value represents the mass of latex, expressed in mg.

[0059] 1.7 Data Analysis Linear regression analysis was performed using SPSS software, and graphs were generated using Origin software. All data are expressed as mean ± standard deviation.

[0060] 2. Test Results 2.1 Results of Fluorescent Dye Parameter Optimization Experiment 2.1.1 Dye concentration and latex concentration The staining time for all three dyes was uniformly set at 90 min, and the results are shown in Table 1. Using Cy5 as the fluorescent dye, both the detectable latex concentration range and the correlation coefficient increased with increasing dye concentration. The correlation coefficient began to decrease after the dye concentration reached 2.0 μM. Therefore, the optimal staining concentration for Cy5 dye was 2.0 μM, with a detectable latex concentration range of 0.2–4.0%. Using AF488 as the fluorescent dye, both the detectable latex concentration range and the correlation coefficient initially increased and then decreased with increasing dye concentration. The optimal staining concentration for AF488 dye was 2.0 μM, with a detectable latex concentration range of 0.4–3.0%. Using AF594 as the fluorescent dye, both the detectable latex concentration range and the correlation coefficient increased with increasing dye concentration. The optimal staining concentration for AF594 dye was 3.0 μM, with a detectable latex concentration range of 0.4–6.0%.

[0061] Table 1. Correlation coefficients of linear regression equations under different dye and latex concentrations. 2.1.2 Staining time The concentrations of both dyes and the latex were determined according to the results in 2.1.1. After a dark reaction of 60 min, measurements were taken every 10 min, and the results are shown in Table 2. Overall, the correlation coefficients of the three dyes showed a trend of first increasing and then decreasing with increasing dyeing time. Specifically, for Cy5 dye, the correlation coefficient R0 was [not specified] during the dyeing period of 60-150 min. 2 All were greater than 0.99; the correlation coefficient R of AF488 dye after 90 min of dyeing was... 2 The correlation coefficient R of AF594 dye at 120 min of dyeing is greater than 0.99. 2 Greater than 0.99. R 2 A value greater than 0.99 indicates a highly significant correlation between fluorescence value and latex protein content. Considering that this method is for rapid detection of latex protein content, the optimal staining time for Cy5 is 60 min, for AF488 it is 90 min, and for AF594 it is 120 min.

[0062] Table 2. Correlation coefficients of linear regression equations under different staining times. 2.1.3 Selection of Fluorescent Dyes Based on the screening results in 2.1.1 and 2.1.2, the regression curves of Cy5 and AF488 dyes are a straight line in Figure 1 (a) and (b); Figure 1 In (c), the regression curve of AF594 dye tends to be a broken line, which is more suitable for detecting latex protein content when the latex concentration is 2.0-6.0% (Table 3). Considering that Cy5 has a shorter staining time, is suitable for a wider range of latex concentrations, and is much cheaper than AF488 and AF594, Cy5 is the preferred fluorescent dye for latex protein detection.

[0063] Table 3. Correlation coefficients of linear regression equations under different fluorescent dye conditions 2.2 Plotting the Standard Curve As can be clearly seen from Figure 2, within the range of latex protein mass of 5.53-55.3 μg, the fluorescence value increases linearly with the increase of latex protein mass. The fitted linear regression equation is Y=48978x+367450, and the correlation coefficient R is [missing value]. 2 The correlation coefficient was 0.997, indicating a highly significant correlation between the two. The mass of the corresponding latex protein in the sample can be calculated based on the fluorescence value reading, thus determining the protein content of the latex.

[0064] 2.3 Detection of latex protein content Table 4 compares the latex protein content measured by the BCA method and the fluorescent labeling detection method. Significance analysis revealed no significant difference in latex protein content between the two methods, indicating that the fluorescent labeling detection method is effective in determining the protein content of rubber tree latex. Furthermore, compared to the BCA method, the fluorescent labeling detection method offers several advantages: it is simple and rapid to operate, uses non-polluting reagents, and is unaffected by reducing agents, making it a highly suitable technique for rapid detection of latex protein content in rubber trees.

[0065] Table 4 Comparison of BCA method and fluorescent labeling detection method for latex protein determination Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

Claims

1. A rapid detection method for protein content in rubber tree latex based on fluorescent probe labeling, characterized in that, Includes the following steps: S1. Sample Selection and Pretreatment: Fresh latex was collected from the field and transported on ice to prevent coagulation. After filtering the latex through a sieve to remove impurities, the latex sample was accurately weighed into a centrifuge tube and diluted with distilled water. The mass of the latex was M. N The unit is mg, and the dilution factor is N; S2. Staining of the sample: Add protein fluorescent dye to the diluted latex obtained in step S1, mix gently and let stand for more than 30 minutes to allow the dye to stain fully. The final volume is V, and the unit is ml. The protein fluorescent dye includes cyanide dye Cy5, CD3 mouse monoclonal antibody and recombinant streptavidin protein. S3. Sample Detection: Aliquot 0.2 mL of the stained latex obtained in step S2 into microplates, perform fluorescence detection using a multi-functional microplate reader, and record the fluorescence value Y. L ; S4. Establishment of standard curve: Select latex protein of different masses as standard samples for protein quantification. Collect fluorescence signals using the same parameters as the sample detection. With the x-axis X as the mass of latex protein and the y-axis Y as the fluorescence value, the linear equation for the mass of latex protein is obtained as Y=aX+b. S5. Calculation of protein mass in latex: Calculate the protein mass in latex using the following formula: M L =(Y L -b) / a, where M L Y represents the mass of latex protein, in μg. L The fluorescence value of the latex to be tested; S6. Calculation of protein content in latex: The protein content of latex is calculated according to the following formula: F = (M L ×N×V) / (M N ×0.2)×100%, where F is the latex protein content, in %; M L The values ​​are: M = (N - V) / (V - M) ... V) = (N - V) / (V - V) = (N - V) / (V - V) = N The value represents the mass of latex, expressed in mg.

2. The rapid detection method for protein content in rubber tree latex according to claim 1, characterized in that, In step S1, accurately weigh 1 mL of latex into a centrifuge tube, with a dilution factor between 50 and 200, and the diluent is distilled water or a weakly alkaline buffer solution; the latex dilution factor is preferably 100, and the diluent is preferably 0.1×PBS buffer.

3. The rapid detection method for protein content in rubber tree latex according to claim 1, characterized in that, In step S2, the preferred protein fluorescent dye is Cy5, with a working concentration of 0.5-3.0 μM and a staining time of 60-150 min; more preferably, the optimal working concentration of Cy5 is 2.0 μM and the optimal staining time is 100 min.

4. The rapid detection method for protein content in rubber tree latex according to claim 1, characterized in that, In step S3, a black bottom permeable microplate is selected, the excitation light is 650nm, the emission light is 670nm, and the operating temperature of the multifunctional microplate reader is 25℃.

5. The rapid detection method for protein content in rubber tree latex according to claim 1, characterized in that, In step S4, the method for preparing the latex protein standard solution includes the following steps: S41. Mix the latex with the protein extraction solvent in the specified ratio and shake to mix thoroughly; S42. Add benzyl sulfonyl fluoride protease inhibitor to the mixture from step S41. S43. Let the mixture from step S42 stand at room temperature for at least 1 hour; S44. The mixture obtained in step S43 is centrifuged at 4°C to separate into layers. S45. Take the intermediate supernatant of the liquid obtained in step S44, place it in a new centrifuge tube, and centrifuge at 4°C to separate the layers. S46. Take the intermediate supernatant of the liquid obtained in step S45 and place it in a new centrifuge tube. This is the latex protein extract. Since the protein extract contains particle detergent, the protein content is detected by BCA method.

6. The rapid detection method for protein content in rubber tree latex according to claim 5, characterized in that, In step S41, the protein extraction solvent is: 7M urea, 2M thiourea, 40 mM Tris, 4% Triton X-100, 0.07% β-mercaptoethanol, and the volume ratio of latex to protein extraction solution is 1:

3. The latex should be added drop by drop, and the mixture should be shaken regularly.

7. The rapid detection method for protein content in rubber tree latex according to claim 5, characterized in that, In step S42, the concentration of PMSF is 100 mM, and the volume ratio of PMSF to the mixture in step S41 is 1:

200.

8. The rapid detection method for protein content in rubber tree latex according to claim 5, characterized in that, In step S43, the settling time is 1-2 hours.

9. The rapid detection method for protein content in rubber tree latex according to claim 5, characterized in that, In step S44, the centrifuge speed is 14000-18000 rpm and the centrifugation time is 45-60 min; The preferred centrifuge speed is 18,000 rpm and the centrifugation time is 60 min.

10. The rapid detection method for protein content in rubber tree latex according to claim 5, characterized in that, In step S45, the centrifuge speed is 14000-18000 rpm, and the centrifugation time is 15-30 min; The preferred centrifuge speed is 14,000 rpm and the centrifugation time is 15 min.

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