Protein quantitative kit widely compatible with components of protein lysate

Abstract

The invention discloses a protein quantitative kit widely compatible with components of a protein lysis solution, and also provides a protein quantitative method widely compatible with components of the protein lysis solution, the method is simple to operate, does not need complex procedures and conditions, also does not need tedious, expensive and complex instruments and equipment, and compared with a Pierce 660 nm protein quantitative kit, the method has the advantages that the detection sensitivity is high, and the detection time is short. The deviation is small and the detection result is more accurate.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a protein quantitative kit which is widely compatible with protein lysate components. Background Art

[0002] Proteomics is a discipline that studies the expression, structure, function and interaction of all proteins in cells, tissues or organisms. Protein extraction is the first step in proteomics experiments, and its purpose is to extract proteins from samples. Protein quantification is a very critical step in proteomics experiments, which aims to determine the protein concentration in the extracted protein sample so as to standardize subsequent experiments (such as gel electrophoresis, mass spectrometry, etc.). There are many common protein quantification methods, each of which has its advantages and disadvantages.

[0003] Laboratories usually use different protein extraction and lysis buffers according to different samples and experimental purposes. Different protein extraction and lysis buffers require different protein quantification kits, and some lysis buffers contain both high-concentration detergents and high-concentration reducing agents. Commonly used kits are incompatible and cannot complete quantification. For example, SDT lysis buffer: SDT lysis buffer (Sodium Dodecyl Sulfate-Temperature Detergent, SDT) is a buffer commonly used for protein extraction and lysis. SDT lysis buffer is usually used to extract total protein from cell or tissue samples, especially for sample preparation in proteomics research. SDT lysis buffer can effectively lyse cell membranes, dissolve most proteins in cells, and protect proteins from degradation. The formula of SDT lysis buffer is as follows: SDS, 1% (w / v) Tris-HCl (pH 7.5), 50 mM DTT, 100 mM Protease inhibitors, and additional reagents as needed (such as PMSF, protease inhibitor cocktail, etc.); Since SDT lysis buffer contains decontaminated SDS and high-concentration DTT (reducing agent), it will have a certain impact on the quantitative method of protein. In the Bradford method, SDS will interfere with the quantitative reagent, and in the BCA method, DTT will interfere with the quantitative reagent, so it is necessary to choose a suitable quantitative method.

[0004] There are many specially designed quantitative kits on the market, especially to solve the influence of SDS or other interfering substances, and provide quantitative solutions for specific sample types. For example, Pierce 660 nm protein quantitative kit, Thermo Fisher's Qubit protein quantitative kit, etc., usually reduce the interference of detergents such as SDS on the measurement results. But the price is high, and the reaction to some proteins may not be as sensitive as traditional methods. Among them, only the Pierce 660 nm protein quantitative kit needs to be paired with IDCR (not sold in China) to be directly compatible with SDT lysis buffer. There are also kit solutions in China that use dialysis or organic solvent precipitation to replace the solution first and then quantify. However, the dialysis operation is complicated and time-consuming, and requires a dedicated dialysis bag or dialysis preparation; after the organic solvent protein is precipitated, it needs to be further dissolved, and the protein may be lost during the precipitation process, and the residue of organic solvent may affect the subsequent analysis. Therefore, providing an economical, easy-to-operate, and widely compatible protein extraction lysis buffer protein quantitative kit is important for proteomics research. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the purpose of the present invention is to provide a protein quantification kit that is widely compatible with protein lysate components.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A first aspect of the present invention provides a protein quantification kit that is widely compatible with protein lysate components.

[0007] Furthermore, the kit includes Sera-Mag SpeedBead magnetic beads, BCA quantitative reagent, organic solvent and protein standard.

[0008] Furthermore, the BCA quantitative reagent includes BCA reagent A solution and BCA reagent B solution.

[0009] Furthermore, the BCA reagent A solution is prepared by mixing 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide and 0.95% sodium bicarbonate, and the pH value is 10-12.

[0010] Furthermore, the BCA reagent B solution is a 4% copper sulfate solution.

[0011] In some embodiments, the pH value of the BCA reagent A solution is adjusted to 10-12 by using a sodium hydroxide solution. In a preferred embodiment, the pH value of the BCA reagent A solution is adjusted to 11.25 by using a sodium hydroxide solution.

[0012] Furthermore, the protein standard comprises a BSA standard.

[0013] Furthermore, the organic solvent includes acetonitrile, ethanol and methanol.

[0014] Preferably, the organic solvent is acetonitrile.

[0015] Furthermore, the kit may also include protein extraction lysis solution and protease inhibitors.

[0016] Furthermore, the protein extraction lysis solution includes SDS lysis solution, RIPA lysis solution, NP-40 lysis solution, Tris-Triton lysis solution, and Tris-HCl lysis solution.

[0017] In the present invention, the kit may include one or more containers containing commercially and user-desirable materials, including water, diluents, and package inserts containing instructions for use. A label may be present on the container to indicate that the reagent is used for protein quantification applications.

[0018] A second aspect of the present invention provides a protein quantification method that is widely compatible with protein lysate components.

[0019] Furthermore, the method includes the following steps: adding the sample to be tested and the organic solvent to a sample tube containing Sera-MagSpeedBead magnetic beads, mixing and incubating at room temperature, and then shaking and incubating. After the shaking and incubation, placing a magnetic stand to remove the supernatant, washing the magnetic beads with an organic solvent, adding water for non-contact ultrasound, and vortex mixing to obtain a protein solution to be tested containing magnetic beads; and then using the BCA method to detect the protein content in the protein solution to be tested.

[0020] Furthermore, the method further comprises the step of washing the Sera-Mag SpeedBead magnetic beads before adding the sample to be tested and the organic solvent into the sample tube containing the Sera-Mag SpeedBead magnetic beads.

[0021] In some embodiments, the washing of the Sera-Mag SpeedBead magnetic beads can be performed using PBS buffer or water. In a specific embodiment of the present invention, the magnetic beads are washed twice using Wahaha water.

[0022] Furthermore, the organic solvent includes acetonitrile, ethanol and methanol.

[0023] Preferably, the organic solvent is acetonitrile.

[0024] Furthermore, the final concentration of acetonitrile is at least 50%.

[0025] In some embodiments, the final concentration of acetonitrile is at least 50%, 60%, 70%, 80%, 90%, or 100%.

[0026] Furthermore, the mixture is incubated at room temperature for 10 to 20 minutes and then incubated with shaking.

[0027] Preferably, the mixing is incubated at room temperature for 15 minutes and then incubated with shaking.

[0028] Furthermore, the shaking incubation is performed using a shaker.

[0029] Preferably, the shaking incubation time is 10 min to 20 min.

[0030] Preferably, the shaking incubation time is 15 min.

[0031] Preferably, the non-contact ultrasound duration is 30 s.

[0032] In a preferred embodiment, Sera-Mag SpeedBead magnetic beads are washed twice with Wahaha water and dispensed into sample tubes, acetonitrile and the sample to be tested are added to the sample tubes, mixed and incubated at room temperature for 15 minutes, and then shaken and incubated. After the shaking and incubation, they are placed on a magnetic stand for 2 minutes, the supernatant is removed, and the magnetic beads are washed 4 times with 200uL 100% acetonitrile, and then 25uL water is added for non-contact ultrasound for 30s, and vortexed to obtain a protein solution to be tested containing magnetic beads; and the protein content in the protein solution to be tested is detected by the BCA method.

[0033] Further, the step of detecting the protein content in the protein solution to be detected by the BCA method comprises: Fitting the standard curve: Mix BCA reagent A solution and BCA reagent B solution to obtain BCA working solution, add BCA working solution to the gradient concentration protein standard, let it stand, and detect the OD value at a wavelength of 562nm respectively. After fitting, the standard curve is obtained; Detection of the protein solution to be detected: Take the BCA working solution and add it to the protein solution to be detected containing magnetic beads, shake and react at 37°C. After the reaction is completed, place it on a magnetic rack, take the supernatant and detect the OD value at a wavelength of 562nm, substitute it into the standard curve, and obtain the protein content in the protein solution to be detected.

[0034] In some embodiments, the volume ratio of the BCA reagent A solution to the BCA reagent B solution is (40-60):1.

[0035] In a preferred embodiment, the volume ratio of the BCA reagent A solution to the BCA reagent B solution is 50:1.

[0036] In some embodiments, the protein standard is prepared by taking a bovine serum albumin (BSA) reference substance or a national standard for protein content determination, dissolving it with 1×PBS or water and diluting it to a final concentration of 0, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, or 2 mg / mL as a protein standard.

[0037] In one embodiment, 20 μL of each of the protein standards diluted to final concentrations of 0, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL in the fitted standard curve are added to the wells of a 96-well plate.

[0038] In one embodiment, in the fitting standard curve, the BCA working solution is added to the protein standard of gradient concentration and then placed at 20-40° C. for 20-120 min. Preferably, 200 μL of BCA working solution is added to the protein standard of gradient concentration and then placed at 37° C. for 30 min.

[0039] In a preferred embodiment, take BCA reagent A solution and B solution and mix them at a volume ratio of 50:1 to obtain BCA working solution. Take 20 μL of each protein standard diluted to a final concentration of 0, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, and 2 mg / mL and add them to a 96-well plate. Add 200 μL of BCA working solution to each reaction well and place it at 37 ° C for 30 min. Measure the absorbance value at A562nm with an enzyme reader.

[0040] In a preferred embodiment, in the detection of the protein solution to be detected, 200 μL BCA working solution is added to the protein solution to be detected containing magnetic beads, and the reaction is shaken at 37° C. for 30 minutes. After the reaction is completed, it is placed on a magnetic stand for 2 minutes, and the supernatant is taken to a 96-well plate. The OD value at a wavelength of 562 nm is detected by an enzyme reader, and the standard curve is substituted to obtain the protein content in the protein solution to be detected.

[0041] Furthermore, the mass ratio of the magnetic beads to the protein is (1-20):25.

[0042] Preferably, the mass ratio of the magnetic beads to the protein is 1:5.

[0043] Furthermore, the sample to be tested includes a protein solution extracted from cells, fresh tissues, blood or urine.

[0044] The third aspect of the present invention provides use of Sera-Mag SpeedBead magnetic beads, the kit described in the first aspect of the present invention, or the method described in the second aspect of the present invention in protein quantification.

[0045] In the present invention, the term "comprising / including" is used to refer to a composition or method of one or more named elements or steps that is open-ended, meaning that the named elements or steps are required, but other elements or steps can be added within the scope of the composition and method. To avoid cumbersomeness, it should also be understood that any composition or method described as "comprising / including" one or more named elements or steps also describes a corresponding more limited composition or method that "essentially consists of the same named elements or steps", meaning that the composition or method includes the named required elements or steps, and may also include other elements or steps that do not substantially affect the basic and novel characteristics of the composition or method.

[0046] In the present invention, the term "agent" used should not be interpreted narrowly, but should be extended to small molecules, protein molecules, such as peptides, polypeptides and proteins, and compositions containing them, as well as genetic molecules, such as RNA, DNA and their mimetics and chemical analogs and cellular agents.

[0047] Advantages and benefits of the present invention: The protein quantification method provided by the present invention is simple to operate, compatible with various protein extraction lysates, and compared with the Pierce 660 nm protein quantification kit, has a small deviation and a more accurate detection result. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Display the electrophoresis results corresponding to different amounts of magnetic beads; Figure 2 Display the saturation curve of gray value corresponding to different amounts of magnetic beads; Figure 3 Shows the standard curve for detecting BSA standards using the BCA method; Figure 4 Display the SDS-page results of magnetic bead-bound proteins; Figure 5 The standard curve of the Pierce 660 nm protein quantitation kit for detecting BSA standards is shown; Figure 6 The detection accuracy of Pierce 660 nm and the method of the present invention is shown; Figure 7 Displays the results of BSA standard protein quantification. DETAILED DESCRIPTION

[0049] 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 clearly and completely described in combination with the embodiments of the present invention. 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.

[0050] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0051] Before conducting the experiment, the applicant analyzed and summarized the advantages and disadvantages of commonly used total protein quantitative analysis methods, as shown in Table 1. Therefore, the purpose of the present invention is to provide a protein quantitative kit that is economical, easy to operate, and widely compatible with protein extraction and lysis solutions.

[0052] Table 1 Advantages and disadvantages of commonly used total protein quantitative analysis methods

[0053] Example 1 Protein Extraction

[0054] 1. Experimental Materials

[0055] HeLa cells, SDT lysis buffer (prepared in the laboratory: 4% SDS, 100mM DTT, 50mM Tris-HCL, pH=8.0), protease inhibitor (MCE HY-K0011), RIPA lysis buffer (MCE HY-K1001).

[0056] 2. Experimental Methods

[0057] SDT lysis protein extraction method:

[0058] 1. Collect the HeLa cell pellet, add 500 µL SDT lysis buffer (add protease inhibitors at 1:50 (v:v) before use, freshly prepared and used), and use a pipette tip to disperse the cells;

[0059] 2. Non-contact ultrasound, ultrasound for 30 seconds, stop for 10 seconds, cumulatively for 20 minutes;

[0060] 3. Place the ultrasonic samples in a pre-cooled low-temperature centrifuge at 4°C, 12,000 g, for 20 min.

[0061] 4. Take the supernatant and divide it into 25µl for quantitative measurement. Store the remaining sample at -80℃ for later use.

[0062] RIPA lysis buffer protein extraction method:

[0063] a. Collect the HeLa cell pellet, add 500 µL RIPA lysis buffer (add protease inhibitors at 1:50 (v:v) before use, freshly prepared and used), and use a pipette tip to disperse the cells;

[0064] b. Non-contact ultrasound, ultrasound for 30 seconds, stop for 10 seconds, cumulatively for 20 minutes;

[0065] c. Place the ultrasonic samples in a pre-cooled low-temperature centrifuge at 4°C, 12,000 g, for 20 min.

[0066] d. Take the supernatant and dilute it 1:10 for Bradford quantification. Dilute the sample to 1µg / uL and store the remaining sample at -80℃ for later use.

[0067] Example 2 Magnetic Bead Loading Test

[0068] 1. Experimental Materials

[0069] Sera-Mag SpeedBead magnetic beads (cytiva 65152105050250).

[0070] 2. Experimental Methods

[0071] 1. Set the magnetic bead amount gradient: 0.1µg, 0.5µg, 1µg, 2µg, 5µg, 10µg, 20µg;

[0072] 2. Calculate and wash beads: Calculate the required amount of magnetic beads, pay attention to adding blank control, wash twice with 1000µL of Wahaha water, divide into sample tubes according to the amount of magnetic beads, adsorb the magnetic beads for 2 minutes, remove the supernatant, and add 5µL of water to resuspend the magnetic beads.

[0073] 3. Then mix 40 µL of ACN and 25 µL of sample (RIPA extracted HeLa cell protein solution, 1 µg / uL) and incubate at room temperature at 1000 rpm for 15 min;

[0074] 4. After the incubation on the shaker, place the tube on a magnetic stand for 2 minutes, remove the supernatant with a pipette, and then wash 4 times with 200 µL 100% acetonitrile;

[0075] 5. Add 10 µl loading buffer, perform non-contact sonication for 30 seconds, and vortex to mix;

[0076] 6. Perform electrophoresis and use image J to calculate the gray value and draw the saturation curve.

[0077] 3. Experimental Results

[0078] The electrophoresis results are as follows Figure 1The grayscale values ​​corresponding to different amounts of magnetic beads are shown in Table 2, and the corresponding saturation curves are shown in Figure 2 These results show that 2µg of magnetic beads can bind 25µg of protein, with 5µg reaching the maximum value. This indicates that the best binding amount is achieved when the magnetic bead-protein ratio is 1:5.

[0079] Table 2 Grayscale values ​​corresponding to different amounts of magnetic beads

[0080]

[0081] Example 3 Drawing of BCA curve

[0082] 1. Experimental Materials

[0083] BCA quantitative reagent (cwbio CW0014S), BSA standard (bovine serum albumin, 2 mg / mL, Thermo23209).

[0084] 2. Experimental Methods

[0085] 1. Dilute the BSA standard: dilute the BSA standard according to Table 3 using the diluent consistent with the protein sample to be tested;

[0086] Table 3 BSA standard dilution

[0087]

[0088] 2. Calculate the total amount of BCA working solution required, mix BCA-A and BCA-B in a volume ratio of 50:1 to prepare BCA working solution, and mix thoroughly;

[0089] 3. According to Table 3, add 25 μL of the diluted AG BSA standard to the marked wells of the 96-well plate. It is recommended to perform 2-3 parallel reactions for each sample to be measured.

[0090] 4. Add 200 μL of BCA working solution to each well, mix thoroughly, cover the 96-well plate, incubate at 37°C for 30 minutes, and cool to room temperature; use an ELISA reader to measure the absorbance of each sample and BSA standard at 562 nm;

[0091] 5. Draw a standard curve.

[0092] 3. Experimental Results

[0093] When using the BCA method for protein quantification, in order to accurately determine the protein concentration in the sample, a set of standard solutions of known concentrations needs to be prepared. After obtaining the absorbance data, the linear regression method can be used to obtain the equation of the standard curve. By substituting the absorbance value of the sample to be tested, the protein concentration of the sample can be calculated. In this embodiment, the absorbance is measured using a BSA standard of known concentration. The measured absorbance values ​​are shown in Table 4, and the standard curve is shown in Table 4. Figure 3 shown.

[0094] Table 4 Absorbance corresponding to different concentrations of BSA standards

[0095]

[0096] Example 4: Using the method of the present invention to quantify protein in a sample to be tested

[0097] 1. Experimental Materials

[0098] Sera-Mag SpeedBead magnetic beads, water, acetonitrile ACN, SDT lysis buffer, SDT-extracted HeLa cell protein solution, and BCA quantitative reagent.

[0099] 2. Experimental Methods

[0100] 1. Calculate and wash beads: Take 10µg beads per sample to calculate the required amount of magnetic beads, add a blank control, wash twice with 100µL of Wahaha water, and dispense into sample tubes;

[0101] 2. Then add 45µL of ACN and 25µL of the sample to be tested (SDT-extracted HeLa cell protein solution) / blank control (SDT lysate) into the sample tube. The final concentration of ACN must be >50%;

[0102] 3. Mix well and incubate at room temperature for 15 minutes;

[0103] 4. Incubate on a shaker for 10-20 minutes, preferably 15 minutes. After incubation, place on a magnetic stand for 2 minutes, remove the supernatant with a spray gun, and then wash 4 times with 200 µL 100% acetonitrile;

[0104] 5. Add 25 µL of water, perform non-contact ultrasound for 30 seconds, and vortex to mix;

[0105] 6. Calculate the total amount of BCA working solution required, mix BCA reagent A solution and B solution at a ratio of 50:1, add 200µL BCA working solution to the magnetic beads treated in the previous step, and shake at 37 degrees Celsius for 30 minutes;

[0106] 7. After the incubation on the shaker, place the cells on a magnetic rack for 2 minutes and transfer the supernatant to an ELISA plate.

[0107] 8. Use an ELISA reader to simultaneously measure the absorbance of the standard and sample at 562 nm;

[0108] 9. Subtract the absorbance value of the blank control from the absorbance value of the sample to be tested to calculate the concentration of the sample to be tested.

[0109] 3. Experimental Results

[0110] The results of protein quantification of the sample to be tested using the method of the present invention are as follows: Figure 4 As shown in Table 5, the SDS-page results of the supernatant during protein binding and washing and the magnetic beads after binding showed that all proteins were bound to the magnetic beads and no protein was lost during the washing process. The measured concentration of the sample Hela-SDT (hela cell protein solution extracted by SDT) was 1.39 mg / mL.

[0111] Table 5 Concentrations of samples measured using the method of the present invention

[0112]

[0113] Example 5: Using the Pierce 660nm Protein Quantitation Kit with IDCR to quantify protein in samples to be tested

[0114] 1. Experimental Materials

[0115] Pierce 660nm protein quantification kit (Cat. No. 22660), IDCR (Cat. No. 22663), BSA standard (bovine serum albumin, 2 mg / mL), SDT lysis buffer, and SDT-extracted HeLa cell protein solution.

[0116] 2. Experimental steps

[0117] 1. Dissolve 1 pack of IDCR in 20 mL of Pierce 660nm protein quantification reagent;

[0118] 2. Dilute the BSA standard according to the instructions;

[0119] 3. Add 10 μL of the diluted BSA standard, the test protein (SDT-extracted HeLa cell protein solution, diluted five times), and the blank sample to the marked 96-well microwells. It is recommended to perform 2-3 parallel reactions for each sample to be measured.

[0120] 4. Add 150 μL of protein quantification reagent to each well, place in the microplate reader and shake at medium speed for 1 minute, and incubate at room temperature for 5 minutes;

[0121] 5. Use an ELISA reader to measure the absorbance of each sample and BSA standard at 660 nm;

[0122] 6. Draw a standard curve and calculate the concentration of the sample to be tested by subtracting the absorbance value of the blank control from the absorbance value of the sample to be tested.

[0123] 3. Experimental Results

[0124] The absorbance of BSA standards of different concentrations at 660 nm is shown in Table 6, and the corresponding standard curve is shown in Figure 5 The results of the Pierce 660nm protein quantification kit with IDCR to detect the concentration of the sample are shown in Table 7, showing that the Hela-SDT concentration detected after five-fold dilution is 0.26 mg / mL, and its original concentration is calculated to be 1.32 mg / mL; compared with the method of the present invention, its detection result is low and inaccurate.

[0125] Table 6 Absorbance of BSA standards at different concentrations at 660 nm

[0126] Table 7 Pierce 660 nm Protein Quantitation Kit with IDCR Detection Results

[0127]

[0128] According to the detection value of Pierce 660 nm protein quantification kit and the kit of the present invention, 10ug was taken for electrophoresis, and the results were as follows Figure 6 As shown, the results show that the detection results of the two methods are similar and can support each other's accuracy.

[0129] Example 6 Protein quantification of diluted BSA standard using Pierce 660 nm protein quantification kit with IDCR and the method of the present invention

[0130] 1. Experimental Materials

[0131] Pierce 660 nm protein quantitation kit (Cat. No. 22660), IDCR (Cat. No. 22663), BSA (250 μg / mL, 125 μg / mL, 62.5 μg / mL), Sera-Mag SpeedBead magnetic beads, water, acetonitrile, SDT lysis buffer, and BCA quantitation reagent.

[0132] 2. Experimental Methods

[0133] 1. Use SDT lysis buffer to dilute BSA standard to final concentrations of 250μg / mL, 125μg / mL, and 62.5μg / mL;

[0134] 2. Detection was performed according to Example 4 (i.e., the method of the present invention) and Example 5 (i.e., the Pierce 660nm method);

[0135] 3. Calculate the detection concentration.

[0136] 3. Experimental Results

[0137] The diluted BSA standard was quantified using the Pierce 660 nm protein quantification kit in combination with IDCR and the method of the present invention. The results are as follows: Figure 7 As shown, it can be seen that the method of the present invention is more accurate in detecting protein concentration and has a smaller deviation.

[0138] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A protein quantification kit that is widely compatible with protein lysate components, characterized in that: The kit comprises Sera-Mag SpeedBead magnetic beads, BCA quantitative reagent, organic solvent and protein standard.

2. The kit according to claim 1, characterized in that The BCA quantitative reagent includes BCA reagent A liquid and BCA reagent B liquid; The BCA reagent A solution is prepared by mixing 1% BCA disodium salt, 2% anhydrous sodium carbonate, 0.16% sodium tartrate, 0.4% sodium hydroxide and 0.95% sodium bicarbonate, with a pH value of 10-12; The BCA reagent B solution is a 4% copper sulfate solution.

3. The kit according to claim 1, characterized in that The protein standard includes a BSA standard; Preferably, the organic solvent comprises acetonitrile, ethanol and methanol; Preferably, the organic solvent is acetonitrile.

4. The kit according to claim 1, characterized in that The kit may also include protein extraction lysis solution and protease inhibitors; Preferably, the protein extraction lysate includes SDS lysate, RIPA lysate, NP-40 lysate, Tris-Triton lysate, or Tris-HCl lysate.

5. A protein quantification method that is widely compatible with protein lysate components, characterized in that: The method comprises the following steps: adding a sample to be tested and an organic solvent into a sample tube containing Sera-Mag SpeedBead magnetic beads, mixing and incubating at room temperature, and then shaking and incubating, placing a magnetic stand after the shaking and incubation to remove the supernatant, washing the magnetic beads with an organic solvent, adding water to perform non-contact ultrasound, and vortex mixing to obtain a protein solution to be tested containing the magnetic beads; and then using the BCA method to detect the protein content in the protein solution to be tested.

6. The method according to claim 5, characterized in that The method further comprises the step of washing the Sera-Mag SpeedBead magnetic beads before adding the sample to be tested and the organic solvent to the sample tube containing the Sera-Mag SpeedBead magnetic beads; Preferably, the organic solvent comprises acetonitrile, ethanol and methanol; Preferably, the organic solvent is acetonitrile; Preferably, the final concentration of acetonitrile is at least 50%; Preferably, the mixing is incubated at room temperature for 10 to 20 minutes and then incubated with shaking; Preferably, the mixing is incubated at room temperature for 15 minutes and then incubated with shaking; Preferably, the shaking incubation is performed using a shaker; Preferably, the shaking incubation time is 10 min to 20 min; Preferably, the shaking incubation time is 15 min; Preferably, the non-contact ultrasound duration is 30 s.

7. The method according to claim 5, characterized in that The step of detecting the protein content in the protein solution to be detected by the BCA method comprises: Fitting the standard curve: Mix BCA reagent A solution and BCA reagent B solution to obtain BCA working solution, add BCA working solution to the gradient concentration protein standard, let it stand, and detect the OD value at a wavelength of 562nm respectively. After fitting, the standard curve is obtained; Detection of the protein solution to be detected: Take the BCA working solution and add it to the protein solution to be detected containing magnetic beads, shake and react at 37°C. After the reaction is completed, place it on a magnetic rack, take the supernatant and detect the OD value at a wavelength of 562nm, substitute it into the standard curve, and obtain the protein content in the protein solution to be detected.

8. The method according to claim 5, characterized in that The mass ratio of the magnetic beads to the protein is (1-20):25; Preferably, the mass ratio of the magnetic beads to the protein is 1:

5.

9. The method according to claim 5, characterized in that The sample to be tested includes protein solution extracted from cells, fresh tissues, blood or urine.

10. Use of Sera-Mag SpeedBead magnetic beads, the kit according to any one of claims 1 to 4, or the method according to any one of claims 5 to 9 in protein quantification.

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