A detergent-compatible bradford protein content assay kit and methods of making and using same

Abstract

The application discloses a detergent-compatible Bradford protein content detection kit and a preparation and use method thereof, and belongs to the technical field of biochemical analysis and detection, and aims to solve the problems of traditional Bradford protein quantification methods, such as intolerance to detergents, easy production of precipitates, leading to distorted detection results, and the problems of existing improved kits, such as complicated operation, poor stability and high cost. The detergent-compatible Bradford protein content detection kit is characterized in that it comprises Bradford quantitative reagents and a series of BSA standard products; the Bradford quantitative reagents comprise coomassie brilliant blue G-250, phosphoric acid, ethanol, oligosaccharide inclusion-type surfactants and deionized water; the series of BSA standard products comprise seven gradient concentrations of BSA aqueous solutions; the application is suitable for rapid quantitative detection of cell and tissue protein samples extracted by various protein lysates containing detergents, and the consistency of detection results of the kit and imported benchmark products is greater than or equal to 95%.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis and detection technology, and relates to a detergent-compatible Bradford protein content detection kit and its preparation and usage method. Specifically, it relates to a ready-to-use detergent-compatible Bradford protein content detection kit and its preparation and usage method, which is suitable for rapid and accurate quantitative detection of cell and tissue protein samples containing detergents. Background Technology

[0002] Protein quantification is one of the most fundamental experimental procedures in molecular biology, biochemistry, and cell biology research. Accurate protein concentration determination is a prerequisite for subsequent experiments such as Western blotting, ELISA, immunoprecipitation, and enzyme activity assays. The Bradford method (Coomassie Brilliant Blue method) is one of the most widely used protein quantification methods. Its principle is that Coomassie Brilliant Blue G-250 dye binds to the basic and aromatic amino acids of proteins under acidic conditions, shifting the dye's maximum absorption peak from 465 nm to 595 nm. The protein concentration can be calculated by detecting the absorbance at 595 nm. This method has advantages such as simple operation, rapid detection, high sensitivity, and compatibility with reducing agents.

[0003] However, the traditional Bradford method has a fatal flaw: it is susceptible to detergent susceptibility. Protein extraction typically requires the addition of detergents such as SDS (sodium dodecyl sulfate), Triton X-100, NP-40 (ethylene glycol ethyl phenyl), and Tween 20 to lyse cells and dissolve proteins. These detergents can bind with Coomassie Brilliant Blue G-250, causing precipitation, resulting in turbidity of the reaction system, abnormally high OD values ​​(optical density values), and severely distorted detection results. To address this problem, existing technologies mainly employ the following two approaches:

[0004] 1. Dilution method: The sample containing detergent is diluted by a high factor to reduce the detergent concentration to a level that does not interfere with the detection. However, this method will reduce the protein concentration, resulting in a decrease in detection sensitivity, and it is not suitable for samples with low protein abundance.

[0005] 2. Modified reagent method: Improve the compatibility of detergents by adjusting the dye formulation or adding precipitation inhibitors, but existing modified products still have obvious shortcomings: some products are only compatible with low concentration detergents (≤0.1%), which cannot meet the needs of conventional protein lysis buffers; some products require the addition of additional clarifying agents or centrifugation steps, which are cumbersome; most imported products are expensive and have poor batch stability.

[0006] Therefore, developing a Bradford protein quantification kit that is compatible with detergents, easy to operate, highly stable, and inexpensive has significant scientific research application value. Summary of the Invention

[0007] The purpose of this invention is to provide a detergent-compatible Bradford protein content assay kit and its preparation and usage method, overcoming the technical defects of the traditional Bradford method being intolerant to detergents and existing improved kits. By adding oligosaccharide-type inclusion surfactants, it achieves tolerance to high concentrations of detergents, and is equipped with ready-to-use standards, simplifying the operation process and improving the accuracy and stability of the assay.

[0008] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0009] In a first aspect, the present invention provides a detergent-compatible Bradford protein content assay kit, comprising a Bradford quantitative reagent and a series of BSA standards; the Bradford quantitative reagent comprises Coomassie Brilliant Blue G-250, phosphoric acid, ethanol, oligosaccharide inclusion surfactants, and deionized water; the series of BSA standards comprises BSA aqueous solutions with seven gradient concentrations.

[0010] The above technical solution effectively shields detergents by adding specific types of oligosaccharide surfactants to the traditional Bradford reagent, utilizing their unique molecular structure to fundamentally solve the detergent interference problem, while retaining the advantages of the traditional Bradford method being fast and simple. The series of BSA (bovine serum albumin) standards includes BSA aqueous solutions with 7 gradient concentrations, which can be used directly without dilution. The Bradford quantitative reagents are independently guaranteed and convenient to use.

[0011] Coomassie Brilliant Blue G-250 is a protein-specific dye. Under acidic conditions, its color changes from brownish-red to blue upon binding with protein, and its absorbance at 595 nm shows a linear relationship with protein concentration. Phosphoric acid provides an acidic reaction environment, ensuring the specific binding of the dye to the protein. Ethanol increases the solubility of Coomassie Brilliant Blue G-250, preventing dye precipitation. The oligosaccharide-type inclusion surfactant is the core innovative component of this invention. It has a unique cyclic molecular structure with a hydrophilic exterior and a hydrophobic interior. Its hydrophobic cavity can bind to the hydrophobic tail of the detergent through van der Waals forces and hydrophobic interactions, forming a stable host-guest inclusion complex. This encapsulates and shields the detergent molecules, preventing non-specific binding and precipitation of Coomassie Brilliant Blue G-250. Simultaneously, this type of surfactant does not interact with proteins or Coomassie Brilliant Blue dye, thus not affecting the specific colorimetric reaction between the dye and protein, ensuring the accuracy of the detection results. A series of BSA standards: Seven pre-prepared standards with varying concentrations are included, eliminating the need for user dilution, reducing operational errors, and improving experimental efficiency.

[0012] Furthermore, based on the total volume of the Bradford quantitative reagent, it contains the following components: Coomassie Brilliant Blue G-250 0.005~0.015 w / v%, phosphoric acid 8~12 v / v%, ethanol 4~8 v / v%, oligosaccharide inclusion surfactant 0.3~0.8 w / v, and the balance being deionized water.

[0013] Furthermore, the concentrations of the series of BSA standards are 0 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL, respectively. Each concentration of standard is individually packaged and stored at -20℃ protected from light.

[0014] Furthermore, the detergent is compatible with Bradford protein assay kits and tolerates SDS, NP-40, Triton X-100, and Tween-20 detergents at concentrations ≤1%. The linear range for protein detection is 100–500 μg / mL, and the linear correlation coefficient R of the standard curve is [value missing]. 2 ≥0.99.

[0015] Furthermore, the oligosaccharide inclusion surfactant is a cyclic oligosaccharide and its derivatives having a hydrophobic inner cavity and a hydrophilic outer surface.

[0016] In the above technical solution, the hydrophobic cavity of the cyclic oligosaccharide and its derivatives with hydrophobic inner cavity and hydrophilic outer surface forms a stable inclusion complex with the detergent molecules, preventing the detergent from binding with Coomassie Brilliant Blue G-250 to produce precipitation, while not affecting the specific binding of Coomassie Brilliant Blue to proteins.

[0017] Furthermore, the cyclic oligosaccharide is composed of 6 to 8 glucose units linked by α-1,4-glycosidic bonds, and its derivatives include any one or more combinations of hydroxyethyl derivatives, hydroxypropyl derivatives, and methylated derivatives.

[0018] Furthermore, the detergent-compatible Bradford protein assay kit is compatible with protein samples including: detergent-containing whole cell protein lysis buffers, nuclear protein lysis buffers, membrane protein lysis buffers, and tissue protein extracts, as well as recombinant protein solutions expressed in prokaryotes and eukaryotes.

[0019] Secondly, the present invention provides a method for preparing the detergent-compatible Bradford protein content assay kit according to any one of the first aspects, comprising the following steps:

[0020] Add phosphoric acid to deionized water and stir until homogeneous to prepare a phosphoric acid aqueous solution;

[0021] Add Coomassie Brilliant Blue G-250 powder to the phosphoric acid aqueous solution and stir until completely dissolved;

[0022] Add ethanol to the Coomassie Brilliant Blue mixture and continue stirring for 10-20 minutes;

[0023] Add oligosaccharide inclusion surfactants to the ethanol mixture and stir for 30-60 minutes until completely dissolved. The solution should be clear and free of precipitate.

[0024] The solution was sterilized by filtration through a ≤0.45 μm filter membrane and stored at 2~8℃ in the dark to obtain the Bradford quantitative reagent;

[0025] BSA stock solution was prepared using 0.9% physiological saline and then sterilized by filtration through a ≤0.45 μm filter membrane.

[0026] The BSA stock solution was serially diluted to prepare a series of BSA standard solutions of 0 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, then aseptically dispensed and stored at -20℃ protected from light.

[0027] By combining Bradford quantitative reagents and a series of BSA standard solutions, a detergent-compatible Bradford protein content assay kit was obtained.

[0028] Thirdly, the present invention provides a method of using the detergent-free Bradford protein assay kit as described in any one of the first aspects, characterized by comprising the following steps:

[0029] Take a 96-well plate and add 10 μL of a series of BSA standards of different concentrations to each well;

[0030] Dilute the protein sample to be tested with PBS or physiological saline, and add 10 μL of each to the well of a 96-well plate.

[0031] Add 300 μL of Bradford quantitative reagent to each of the standard and sample wells, mix gently, and incubate at 10-30°C in the dark for 3-5 minutes.

[0032] The absorbance of each well was measured at a wavelength of 595 nm using an ELISA reader, with the 0 μg / mL standard wells serving as blank controls.

[0033] A standard curve was plotted with the protein concentration of a series of BSA standards on the x-axis and the absorbance value on the y-axis. The protein concentration was calculated based on the absorbance value of the sample and multiplied by the dilution factor to obtain the original sample concentration.

[0034] Furthermore, when using a spectrophotometer to detect protein content, the reaction system was expanded to a volume ratio of 1:30 between the protein sample and the Bradford quantitative reagent, while the detection wavelength remained at 595 nm.

[0035] Beneficial effects

[0036] Excellent detergent compatibility: It can tolerate a variety of commonly used detergents such as 1% SDS, 1% NP-40, 1% Triton X-100, and 1% Tween-20, covering the detergent concentration range of most protein lysis buffers on the market, allowing for direct detection without sample dilution;

[0037] The detection is accurate and reliable: the linear range of the standard curve is 100~500 μg / mL, and the linear correlation coefficient R is [value missing]. 2 ≥0.99; the actual sample test results are ≥95% consistent with the imported benchmark products;

[0038] Simple and quick to operate: Equipped with ready-to-use BSA standards, eliminating the need for user preparation and dilution; readings can be obtained after 3-5 minutes of incubation at room temperature, and the entire testing process takes no more than 15 minutes.

[0039] Excellent stability: Bradford quantitative reagent can be stably stored at 2~8℃ for 12 months without precipitation; accelerated at 37℃ for 14 days, there is no significant change in detergent compatibility and detection performance;

[0040] Low cost: The core raw materials are all domestically produced and readily available reagents. The cost of industrial-scale mass production is only 1 / 3 to 1 / 4 of that of imported similar products, which greatly reduces scientific research costs.

[0041] Wide range of applications: Compatible with various protein samples containing detergents, including whole cell proteins, nuclear proteins, membrane proteins, tissue proteins, and recombinant proteins, suitable for multiple fields such as basic scientific research and drug development. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a comparison of the standard curves of the reagent kit of Example 1 of the present invention under conditions containing 1% SDS, 1% Triton X-100, and 1% NP-40 detergent.

[0044] Figure 2 The graph shows the detergent compatibility verification results of the reagent kit of Example 1 of the present invention after 7 days and 14 days of accelerated cleaning at 37°C. Detailed Implementation

[0045] The above content is further illustrated below with specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments. All technologies implemented based on the above content of this invention fall within the scope of this invention.

[0046] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.

[0047] Example 1: Standardized preparation process of a detergent-compatible Bradford protein content assay kit

[0048] 1. Preparation of Bradford quantitative reagent

[0049] (1) Take 800 mL of deionized water and add it to a 2 L beaker. Slowly add 100 mL of 85% phosphoric acid while stirring to avoid local overheating.

[0050] (2) Accurately weigh 0.1 g of Coomassie Brilliant Blue G-250 powder, add it to the above-mentioned phosphoric acid aqueous solution, and stir magnetically for 30 minutes until the dye is completely dissolved;

[0051] (3) Add 50 mL of 95% ethanol and continue stirring for 15 minutes;

[0052] (4) Accurately weigh 5 g of oligosaccharide inclusion surfactant, wherein the oligosaccharide is a cyclic oligosaccharide composed of 6 to 8 glucose units linked by α-1,4-glycosidic bonds, add it to the mixture, and stir magnetically for 45 minutes until completely dissolved. The solution is a clear brownish-red color.

[0053] (5) Add deionized water to a final volume of 1 L and stir well;

[0054] (6) Use a 0.45 μm microporous membrane for filtration to remove insoluble impurities;

[0055] (7) Dispense into 250 mL brown reagent bottles to obtain Bradford quantitative reagent. Store at 2~8℃ in a dark, sealed container. Shelf life is 12 months.

[0056] 2. Preparation of a series of BSA standards

[0057] (1) Accurately weigh 100 mg of BSA standard, dissolve it in 0.9% sterile physiological saline and make up to 100 mL to prepare a 1 mg / mL BSA stock solution;

[0058] (2) Sterilize by filtration using a 0.22 μm sterile filter membrane;

[0059] (3) The mother liquor was serially diluted with 0.9% sterile physiological saline to prepare standard solutions of 0, 100, 150, 200, 300, 400, and 500 μg / mL;

[0060] (4) Aseptically dispensed into 1.5 mL cryovials, 1 mL per tube, to obtain ready-to-use series of BSA standards;

[0061] (5) Store at -20℃ in a sealed container away from light. Shelf life is 12 months. Avoid repeated freeze-thaw cycles.

[0062] 3. By combining Bradford quantitative reagents and ready-to-use BSA standards, a detergent-compatible Bradford protein content assay kit is obtained.

[0063] Example 2: Instructions for using the detergent-compatible Bradford protein assay kit

[0064] This embodiment uses the 96-well plate method with a detergent-compatible Bradford protein assay kit.

[0065] 1. Experimental preparation: Remove the Bradford quantitative reagent in advance, restore it to 25°C, and mix by inverting the container; remove the series of BSA standards from -20°C, thaw them on ice, and mix thoroughly.

[0066] 2. Sample addition: Take a 96-well plate and add the standard and the sample to be tested according to Table 1. Perform 3 replicates for each sample.

[0067] 3. Incubation: Gently shake the 96-well plate to mix, and incubate at 25°C in the dark for 3-5 minutes;

[0068] 4. Detection: The absorbance of each well was measured at a wavelength of 595 nm using an ELISA reader;

[0069] 5. Result Calculation:

[0070] (1) Plot a standard curve with the concentration of the standard as the abscissa and the corresponding absorbance value (minus the absorbance of the blank well) as the ordinate to obtain the linear regression equation;

[0071] (2) Substitute the absorbance values ​​of the sample wells (minus the absorbance of the blank wells) into the regression equation to calculate the protein concentration of the sample;

[0072] (3) Original sample protein concentration = calculated concentration × sample dilution factor.

[0073] A standard curve was plotted under conditions without detergent. The results showed a linear range of 100–500 μg / mL, with a regression equation of y = 0.0007x - 0.0064 and R0. 2 =0.9955, which meets the testing requirements.

[0074] Table 1: Sample loading amount for 96-well plate.

[0075]

[0076] Example 3: Detergent compatibility verification with the Bradford Protein Content Assay Kit

[0077] This embodiment uses the same method as Example 2 to determine the standard curve, except that 0.25%, 0.5%, and 1% of SDS, Triton X-100, and NP-40 detergents are added to the series of BSA standards respectively, and the standard curve is plotted using the kit of the present invention.

[0078] The experimental results are shown in Table 2. A comparison of the standard curves under conditions containing 1% SDS, 1% Triton X-100, and 1% NP-40 detergents is shown in the figure. Figure 1 As shown, the linear correlation coefficient R of the standard curve is [value missing] for all the detergent concentrations mentioned above. 2 All values ​​are ≥0.99, indicating that the kit of the present invention can effectively tolerate 1% concentration of commonly used detergents without precipitation and with good detection linearity.

[0079] Table 2: Standard curves of BSA standards after adding 0.25%, 0.5%, and 1% SDS, Triton X-100, and NP-40 detergents.

[0080]

[0081] Example 4: Verification of the detergent-compatible Bradford protein assay kit using actual protein samples.

[0082] This embodiment uses the same method as Example 2 to determine the protein sample content. The difference is that the protein samples in the sample wells are Lysis buffer (lysis buffer), RIPA (strong) (radioimmunoprecipitation lysis buffer, strong), and Western and IP protein lysis buffers (immunoblotting and immunoprecipitation protein lysis buffers) to extract total protein from A549, MDA-MB-231, and HepG2 cells. The experimental results are shown in Table 3.

[0083] Example 5: Accelerated stability verification of detergent compatible with Bradford protein assay kit

[0084] The Bradford quantitative reagent from the detergent-compatible Bradford protein content assay kit of this invention was placed in a 37°C incubator for accelerated storage for 7 and 14 days, respectively, to verify the detergent compatibility of Example 3 and to test actual samples in Example 4.

[0085] The results of the detergent compatibility verification after 7 and 14 days of accelerated testing at 37℃ are shown in the figure below. Figure 2 As shown, after 7 and 14 days of accelerated treatment at 37℃, the standard curves R of the reagent kit under conditions containing 1% of various detergents were obtained. 2 The value remained ≥0.99, and the relative deviation between the actual sample test results and the non-accelerated group was ≤3%, indicating that the kit has excellent stability and can be stored for a long time.

[0086] Table 3. Results of reagent stability tests after 7 and 14 days of accelerated testing at 37℃.

[0087]

[0088] Comparative Example 1: Imported benchmark product kit for determining the content of actual protein samples

[0089] The testing method for this comparative example is the same as that in Example 4, except that the protein content detection kit used is the Pierce™ detergent-compatible Bradford assay kit (Pierce, USA, 23246). The experimental results are shown in Table 3. The detection results of the kit of this invention are highly consistent with those of imported products, with relative deviations all ≤5%, indicating that the detection results are accurate and reliable.

[0090] Table 4: Detection results of actual protein samples.

[0091]

[0092] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detergent-compatible Bradford protein assay kit, characterized in that, It includes Bradford quantitative reagents and a series of BSA standards; the Bradford quantitative reagents include Coomassie Brilliant Blue G-250, phosphoric acid, ethanol, oligosaccharide inclusion surfactants, and deionized water; the series of BSA standards includes BSA aqueous solutions with seven gradient concentrations.

2. The detergent-compatible Bradford protein content assay kit according to claim 1, characterized in that, Based on the total volume of the Bradford quantitative reagent, it contains the following components: Coomassie Brilliant Blue G-250 0.005~0.015 w / v%, phosphoric acid 8~12 v / v%, ethanol 4~8 v / v%, oligosaccharide inclusion surfactant 0.3~0.8 w / v, and the balance being deionized water.

3. The detergent-compatible Bradford protein content assay kit according to claim 1, characterized in that, The series of BSA standards have concentrations of 0 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, and 500 μg / mL. Each concentration of standard is individually packaged and stored at -20℃ protected from light.

4. The detergent-compatible Bradford protein content assay kit according to claim 1, characterized in that, The detergent is compatible with Bradford protein assay kits and tolerates SDS, NP-40, Triton X-100, and Tween 20 detergents at concentrations ≤1%. The linear range for protein detection is 100–500 μg / mL, and the linear correlation coefficient of the standard curve is R0. 2 ≥0.

99.

5. The detergent-compatible Bradford protein assay kit according to claim 1, characterized in that, The oligosaccharide-type inclusion surfactant is a cyclic oligosaccharide and its derivatives having a hydrophobic inner cavity and a hydrophilic outer surface.

6. The detergent-compatible Bradford protein assay kit according to claim 1, characterized in that, The cyclic oligosaccharide is composed of 6 to 8 glucose units linked by α-1,4-glycosidic bonds, and its derivatives include any one or more combinations of hydroxyethyl derivatives, hydroxypropyl derivatives, and methylated derivatives.

7. The detergent-compatible Bradford protein assay kit according to claim 1, characterized in that, The detergent-compatible Bradford protein assay kit is compatible with the following protein samples: detergent-containing whole cell protein lysis buffer, nuclear protein lysis buffer, membrane protein lysis buffer, and tissue protein extract, as well as recombinant protein solutions expressed in prokaryotes and eukaryotes.

8. A method for preparing a detergent-compatible Bradford protein assay kit according to any one of claims 1-7, characterized in that, Includes the following steps: Add phosphoric acid to deionized water and stir until homogeneous to prepare a phosphoric acid aqueous solution; Add Coomassie Brilliant Blue G-250 powder to the phosphoric acid aqueous solution and stir until completely dissolved; Add ethanol to the Coomassie Brilliant Blue mixture and continue stirring for 10-20 minutes; Add oligosaccharide inclusion surfactants to the ethanol mixture and stir for 30-60 minutes until completely dissolved. The solution should be clear and free of precipitate. The solution was sterilized by filtration through a ≤0.45μm filter membrane and stored at 2~8℃ in the dark to obtain the Bradford quantitative reagent; BSA stock solution was prepared using 0.9% physiological saline and then sterilized by filtration through a ≤0.45μm filter membrane; The BSA stock solution was serially diluted to prepare a series of BSA standard solutions of 0 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, then aseptically dispensed and stored at -20℃ protected from light. By combining Bradford quantitative reagents and a series of BSA standard solutions, a detergent-compatible Bradford protein content assay kit was obtained.

9. A method of using the detergent-free Bradford protein assay kit according to any one of claims 1-7, characterized in that, Includes the following steps: Take a 96-well plate and add 10 μL of a series of BSA standards of different concentrations to each well; Dilute the protein sample to be tested with PBS or physiological saline, and add 10 μL of each to the well of a 96-well plate. Add 300 μL of Bradford quantitative reagent to each of the standard and sample wells, mix well, and incubate at 10-30°C in the dark for 3-5 minutes; The absorbance of each well was measured at 595 nm using an ELISA reader, with 0 μg / mL standard wells used as blank controls. A standard curve was plotted with the protein concentration of a series of BSA standards on the x-axis and the absorbance value on the y-axis. The protein concentration was calculated based on the absorbance value of the sample and multiplied by the dilution factor to obtain the original sample concentration.

10. The method of using the detergent-free Bradford protein assay kit according to claim 9, characterized in that, When using a spectrophotometer to detect protein content, the reaction system is expanded to a volume ratio of 1:30 between the protein sample and Bradford quantitative reagent, while the detection wavelength remains at 595 nm.

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