Device and method for extracting mitochondrial protein

Abstract

The invention provides a device and a method for extracting mitochondrial protein, and relates to the technical field of biomedicine. According to the method disclosed by the invention, impurities such as plasma in the blood are separated in advance before lysis, meanwhile, the lysis buffer solution with specific components is utilized, the lysis buffer solution is utilized for lysis for a specific time, and meanwhile, mitochondria is crushed in combination with an ultrasonic condition of a specific condition, so that the mitochondrial protein can be efficiently and stably extracted from the human blood. The method disclosed by the invention is standard in operation and rigorous in process, contributes to improving the quality and efficiency of mitochondrial protein extraction, provides a reliable experimental basis for subsequent related research and application, and has important significance in the fields of biomedical research, disease diagnosis and the like. The device provided by the invention can further reduce experimental pollution and errors and improve the extraction quality and accuracy.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a device and method for extracting mitochondrial protein. Background Art

[0002] Mitochondrial proteins play a key role in the physiological processes of cell energy metabolism, signal transduction and apoptosis, and are of great significance to the normal operation of cells. For example, proteins in the mitochondrial respiratory chain work together to store energy, transmit information to regulate cell activities during signal transduction, and key proteins in the apoptosis process affect the apoptotic program.

[0003] However, there are many problems with the current mitochondrial protein extraction technology. On the one hand, the layout of instruments and equipment is scattered. Extraction requires a variety of instruments, such as centrifuges, pipettes, homogenizers, ultrasonic disruptors, etc., which are distributed in different areas of the laboratory. Operators need to transfer samples frequently during sample processing, which makes the samples susceptible to contamination. The mixing of tiny impurities may interfere with subsequent protein analysis, resulting in data deviations or errors. Moreover, the residues and spills during sample transfer cause losses, affecting the quality and accuracy of the extraction, and thus misleading the study of mitochondrial protein function and disease mechanisms. On the other hand, some instruments have poor operating characteristics themselves. The speed control of some centrifuges is inaccurate, which affects the separation of cell components and the enrichment of mitochondrial proteins; the accuracy deviation of the pipette interferes with the volume of reagent addition, affecting the intracellular environment and reaction; improper operation of the homogenizer causes uneven cell disruption, affecting mitochondrial release and protein extraction; the power and time control of the ultrasonic disruptor are not accurate, which can easily cause protein denaturation or incomplete release. These problems restrict the development of mitochondrial protein extraction technology, and improved methods are urgently needed. Summary of the invention

[0004] The embodiments of the present invention provide a device and method for extracting mitochondrial protein to solve at least one of the problems existing in the related art. The present invention aims to provide an optimized method for extracting mitochondrial protein, overcome the deficiencies in the prior art, improve the extraction efficiency of mitochondrial protein, ensure that the effect of the extraction process on the protein structure and function is minimized, and provide high-quality experimental materials for scientific research and clinical application related to mitochondrial protein.

[0005] To achieve the object, the present invention is implemented through the following technical solutions.

[0006] A method for extracting mitochondrial protein comprises the following steps:

[0007] S1, centrifuge peripheral blood, collect precipitate and obtain blood cells;

[0008] S2. Add lysis buffer to the blood cells obtained in step S1, mix well, let stand, repeat mixing and standing, the total standing time is 15-20 minutes, to ensure that the red blood cells are fully lysed; then, centrifuge, collect the precipitate, add lysis buffer to the precipitate, mix well, let stand, repeat mixing and standing, the total standing time is 9-10 minutes, centrifuge, and collect the white blood cell precipitate;

[0009] The lysis buffer comprises water, 140-150 mmol / L ammonium chloride, 0.8-1.2 mmol / L EDTA K2 and 0.8-1.2 mmol / L sodium bicarbonate, with a pH value of 8.0;

[0010] S3, the leukocyte precipitate obtained in step S2 is washed and purified with physiological saline to obtain a purified leukocyte precipitate;

[0011] S4. Add the leukocyte resuspension solution to the leukocyte precipitate purified in step S3, mix well, and homogenize in an ice bath to fully disrupt the leukocytes; fully collect the homogenized leukocyte homogenate (i.e., the solution containing disrupted leukocytes), centrifuge, collect the supernatant, centrifuge, collect the precipitate, add phosphate buffer to the precipitate, mix well, and obtain a mitochondrial solution;

[0012] The composition of the leukocyte resuspension solution is: water, 0.1-0.2 g / L digitonin, 20-25 mmol / L sucrose, 75-80 mmol / L mannitol, 1-1.5 mmol / L EDTA K2 and 10-12 mmol / L Tris (pH 7.2);

[0013] The phosphate buffer is composed of: water, 50-55 mM potassium dihydrogen phosphate and 0.1-0.2 mol / L NaOH;

[0014] S5. In an ice bath state, the mitochondrial solution obtained in step S4 is disrupted by an ultrasonic disruptor to obtain a mitochondrial protein solution.

[0015] Preferably, in steps S1 and S2, the centrifugation conditions are both 2500-3000 rpm for 5-8 minutes.

[0016] Preferably, in steps S1 and S2, the centrifugation condition is 2800 rpm for 6 minutes.

[0017] Preferably, in step S2, the repeated mixing and letting stand is repeated mixing and letting stand for 1-3 minutes each.

[0018] Preferably, in step S2, the repeated mixing and letting stand is repeated for 3 minutes.

[0019] Preferably, in step S2, the lysis buffer comprises: water, 144 mmol / L ammonium chloride, 1 mmol / L ETAK2 and 1 mmol / L sodium bicarbonate, with a pH value of 8.0.

[0020] Preferably, in step S3, the cleaning is performed at least twice.

[0021] Preferably, in step S4, the homogenization is performed at least 20 times.

[0022] Preferably, in step S4, the sufficient collection includes collecting the leukocyte homogenate remaining in the homogenizer.

[0023] Preferably, in step S4, the leukocyte homogenate is centrifuged at 2500-3000 rpm for 10-15 minutes.

[0024] Preferably, in step S4, the leukocyte homogenate is centrifuged at 2800 rpm for 10 minutes.

[0025] Preferably, in step S4, the supernatant is centrifuged at 10600-11000 rpm for 10-15 minutes.

[0026] Preferably, in step S4, the supernatant is centrifuged at 10600 rpm for 10 minutes.

[0027] Preferably, in step S4, the composition of the leukocyte resuspension is: water, 0.1 g / L digitonin, 25 mmol / L sucrose, 75 mmol / L mannitol, 1 mmol / L EDTA K2 and 10 mmol / L Tris (pH 7.2); the composition of the phosphate buffer is: water, 50 mM potassium dihydrogen phosphate and 0.1 mol / L NaOH.

[0028] Preferably, in step S5, the conditions for the ultrasonic crusher to crush are: power 50%, working time 1 min, ultrasonic time 2-4 s, and intermittent time 2-4 s. Preferably, in step S5, the conditions for the ultrasonic crusher to crush are: power 50%, working time 1 min, ultrasonic time 2 s, and intermittent time 2 s.

[0029] A device for extracting mitochondrial protein, the device is an integrated experimental cabin, the integrated experimental cabin comprises an experimental system (1), a lighting system (2) and a ventilation system (3); the experimental system (1) can provide a space and instruments for conducting experiments, the lighting system (2) can provide uniform and appropriate light, and the ventilation system (3) can adjust the air flow and humidity in the cabin to maintain the stability of the experimental environment; the experimental system (1) comprises: a reagent preparation area (11), a sample processing area (12) and a detection area (13), and the areas are interconnected through a sealed channel (14) and an automatic isolation door (15); the reagent A sample transfer window (16) and a high-efficiency air filtration system (17) are arranged between the preparation area (11) and the sample processing area (12), and between the sample processing area (12) and the detection area (13). The sample transfer window (16) is equipped with an automatic opening and closing device and a sample fixing fixture. The high-efficiency air filtration system (17) can effectively prevent air cross contamination between various areas. A buffer room (18) is arranged at the entrance of each area of ​​the reagent preparation area (11), the sample processing area (12) and the detection area (13). A biological safety cabinet (19) is also arranged at the entrance of each area of ​​the reagent preparation area (11) and the sample processing area (12).

[0030] Preferably, the lighting system (2) and the ventilation system (3) are arranged on the top of the integrated experimental cabin.

[0031] Preferably, the reagent preparation area (11) is provided with instruments and equipment used for reagent preparation, including an intelligent reagent storage cabinet, a high-precision electronic balance, an automatic stirrer, an intelligent temperature control system, a disinfection device (ultraviolet lamp, etc.) and a laboratory table. The intelligent reagent storage cabinet has multiple layers of storage racks, each of which is equipped with an independent temperature and humidity sensor and a regulating device, which can accurately control the storage environment of the reagent. The high-precision electronic balance can be accurate to 0.0001g and is used to accurately weigh the reagent components. The automatic stirrer can fully stir the reagent according to the preset stirring speed and time. The intelligent temperature control system can adjust the reagent to the required precise temperature and maintain the temperature stability.

[0032] Preferably, the sample processing area (12) is provided with instruments and equipment for sample processing, including a multifunctional centrifuge, an intelligent vortex instrument, an automated ultrasonic crusher, a Dounce homogenizer and supporting fixtures, disinfection equipment (ultraviolet lamp, etc.) and a laboratory table. The multifunctional centrifuge has a variety of centrifugal modes and adjustable centrifugal force, and its centrifuge tube slot can adapt to centrifuge tubes of different specifications, and an elastic buffer pad and a sample positioning device are provided inside the slot, which can effectively prevent the centrifuge tube from breaking and the sample from shaking. The intelligent vortex instrument can vortex the sample according to the preset vortex speed and time, and has an automatic stop function. The automated ultrasonic crusher has adjustable ultrasonic power, frequency and time, and its ultrasonic probe is positioned by a high-precision mechanical arm, which can accurately control the position and range of ultrasonic crushing, and the surface of the ultrasonic probe is also coated with a special anti-corrosion and anti-pollution coating. The Dounce homogenizer and supporting fixture are made of special materials and designs, which can more effectively crush cells, and the supporting fixture has a clamping and loosening function, and can be automatically adjusted according to the size of the homogenizer.

[0033] Preferably, the detection area (13) is provided with instruments and equipment for detecting the experimental results, including a high-precision protein concentration detection kit, an ELISA reader and a data processing system. The high-precision protein concentration detection kit can quickly and accurately detect the concentration of mitochondrial proteins; the data processing system can analyze, record and store the detection data in real time, and can transmit and share data with external devices.

[0034] The method of using the device of the present invention is as follows: before the experiment, the lighting system (2), the ventilation system (3) and the high-efficiency air filtration system (17) are turned on, and the experimenter enters the experimental cabin and then enters each area through the buffer room (18) at the entrance of the reagent preparation area (11), the sample processing area (12) and the detection area (13).

[0035] First, in the reagent preparation area (11), select, weigh, stir, and adjust the temperature of the reagents in strict accordance with the operating requirements of the reagent preparation stage. When selecting reagents in the intelligent reagent storage cabinet, pay attention to the validity period and storage conditions of the reagents to ensure that the reagents used meet the requirements. When using a high-precision electronic balance to weigh the reagents, place the balance in a stable position to avoid vibration that affects the weighing accuracy. When stirring the reagents, set the stirring speed and time reasonably according to the properties and requirements of the reagents to ensure that the reagents are fully stirred.

[0036] Before entering the experimental cabin, the experimenter should change into a special lab coat, make sure the cuffs are tied tightly, put on gloves on both hands (gloves must completely cover the wrists), and wear a protective mask. Turn on the ultraviolet light in the experimental cabin half an hour in advance for comprehensive disinfection. After disinfection is completed, use 75% alcohol to carefully wipe the surface of the test bench. Specifically, the experimenter enters the experimental cabin and first prepares the sample to be tested (collected peripheral blood sample), physiological saline, white blood cell resuspension, phosphate buffer (pH7.4), lysis buffer, standard bovine serum albumin gradient dilution solution and other reagents in the reagent preparation area (11).

[0037] Next, the sample to be tested and other reagents are transferred to the sample processing area (12) through the sample transfer window (16) between the reagent preparation area (11) and the sample processing area (12), the reagents are fixed by the sample fixing clamp, the automatic opening and closing device is opened, and the reagents are transferred to the sample processing area (12) through the sample transfer window (16). The sample to be tested is processed in the sample processing area (12), and the protein in the peripheral blood mitochondria is extracted to obtain a mitochondrial protein solution. During the sample processing stage, the operator needs to pay close attention to the processing of the sample in each instrument. During the centrifugation process, check whether the centrifuge tube is placed correctly and whether the centrifugal force is appropriate to avoid sample loss or contamination due to improper centrifugation. During the vortexing, ultrasonic disruption and homogenization operations, the operation is performed according to the preset parameters to ensure the accuracy and effectiveness of the operation.

[0038] When transferring samples, operate through the sample transfer window and be careful to keep the sample transfer window clean and closed to prevent the sample from being contaminated.

[0039] As a specific embodiment, the sample processing includes the following steps:

[0040] S1. Centrifuge the peripheral blood sample at 2800 rpm for 6 minutes. After centrifugation, discard the upper plasma to obtain blood cells.

[0041] S2. Add lysis buffer to the obtained blood cells, mix well, and let stand for 15 minutes. During this period, gently shake once every 3 minutes to ensure that the red blood cells are fully lysed. Then centrifuge at a speed of 2800rpm for 6 minutes, discard the supernatant, add lysis buffer to the obtained precipitate, mix well and let stand for 9 minutes, shake once every 3 minutes, and then centrifuge at a speed of 2800rpm for 6 minutes, discard the supernatant, and obtain a white blood cell precipitate. The composition of the lysis buffer is: water, 144mmol / L ammonium chloride, 1mmol / LEDTA K2 and 1mmol / L sodium bicarbonate.

[0042] S3. The obtained leukocyte precipitate was washed and purified with physiological saline, and after washing, it was centrifuged at 2800 rpm for 6 minutes, and the supernatant was discarded to obtain a purified leukocyte precipitate.

[0043] S4. Add leukocyte resuspension to the purified leukocyte precipitate, mix well, and then homogenize 20 times in an ice bath to fully break the leukocytes. Collect the homogenized leukocyte homogenate (i.e., the solution containing broken leukocytes) and centrifuge at 2800rpm for 10 minutes to collect the supernatant. Centrifuge the collected supernatant at 10600rpm for 10 minutes, discard the supernatant, and the precipitate is the mitochondria. Add phosphate buffer to the precipitate and mix well to obtain a mitochondrial solution. The composition of the leukocyte resuspension is: water, 0.1g / L digitonin, 25mmol / L sucrose, 75mmol / L mannitol, 1mmol / L EDTA K2 and 10mmol / LTris (pH7.2); the composition of the phosphate buffer is: water, 50mM potassium dihydrogen phosphate and 0.1mol / L NaOH.

[0044] S5. In an ice bath, the mitochondrial solution is broken by an ultrasonic disruptor, with the power set to 50%, the working time to 1 min, the ultrasonic time to 2 s, the interval time to 2 s, and the mitochondria are broken by ultrasonic waves to obtain a mitochondrial protein solution.

[0045] Finally, the processed sample (mitochondrial protein solution) is transferred to the detection area (13) through the sample transfer window (16) between the sample processing area (12) and the detection area (13), the sample is fixed by a sample fixing fixture, the automatic opening and closing device is opened, and the sample is transferred to the detection area (13) through the sample transfer window (16). The sample is used in the detection area (13) to obtain detection data using a test kit and a detection device, and the data is analyzed to obtain a detection result.

[0046] During the testing phase, when the operator uses the kit to test the extracted mitochondrial protein solution, he / she should ensure that the kit solution is in good condition and the test data is accurate. After obtaining the test data, it is ensured that it is analyzed, recorded and stored in a timely manner, and the operator can view the relevant data as needed.

[0047] The embodiments of the present invention have the following beneficial effects:

[0048] 1. Improve extraction efficiency

[0049] By optimizing the reagent formula and extraction steps, separating blood cells first and then adding lysis buffer, using lysis buffer with specific components and a specific lysis time, and combining multiple centrifugation and washing steps, mitochondria can be more effectively separated from blood, thereby increasing the amount of mitochondrial protein extracted;

[0050] 2. Protect protein structure and function

[0051] During the entire extraction process, the experimental conditions were strictly controlled, such as low temperature operation and avoiding vigorous stirring. Especially in the ultrasonic disruption step, by optimizing the ultrasonic parameters combined with ice bath cooling, the denaturation and degradation of the protein were effectively reduced, ensuring the structural integrity and biological activity of the mitochondrial protein. This is of great significance for subsequent protein function research and drug development based on mitochondrial proteins.

[0052] 3. Easy to operate

[0053] The experimental equipment and reagents involved in the method of the present invention are all routine laboratory equipment, and the operation steps are relatively simple and clear, easy to master and repeat. No special instruments and complex operating skills are required, which reduces the experimental cost and operating difficulty, and is easy to promote and apply in various laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0055] Figure 1 This is the standard curve of Example 1.

[0056] Figure 2 This is a schematic diagram of the structure of the integrated experimental cabin of Example 2.

[0057] Figure 3 This is the experimental system (1) of the integrated experimental chamber of Example 2.

[0058] Reference numerals:

[0059] (1) Experimental system, (2) Lighting system, (3) Ventilation system, (11) Reagent preparation area, (12) Sample processing area, (13) Detection area, (14) Sealed channel, (15) Automatic isolation door, (16) Sample transfer window, (17) High-efficiency air filtration system, (18) Buffer room, (19) Biological safety cabinet. DETAILED DESCRIPTION

[0060] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are proposed in order to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented. The division of the following embodiments is for the convenience of description, and should not constitute any limitation on the specific implementation of the present invention. The various embodiments can be combined and referenced with each other without contradiction.

[0061] NaCl was purchased from Solebao (Sinopharm Group) with the catalog number S8210 (catalog number 10001508). Digitonin was purchased from Solebao with the catalog number D8920; sucrose was purchased from Solebao with the catalog number S8271; mannitol was purchased from Solebao with the catalog number M8141; EDTA K2 was purchased from Solebao with the catalog number E8651; Tris was purchased from Solebao with the catalog number T8060. Potassium dihydrogen phosphate was purchased from Solebao with the catalog number P7392. EDTA K2 was purchased from Solebao with the catalog number E8651; sodium bicarbonate was a product of Sinopharm Group with the catalog number 10018960.

[0062] Physiological saline: Accurately weigh 9 g of NaCl (molecular weight: 58.44), slowly add it to an appropriate amount of deionized water, stir with a magnetic stirrer until completely dissolved, and then dilute to 1 L with deionized water to obtain a physiological saline solution with a concentration of 0.90%.

[0063] Leukocyte resuspension: Weigh 0.01g digitonin (molecular weight 1229.31, concentration 0.1g / L), 0.85575g sucrose (molecular weight 342.3, target concentration set to 25mmol / L), 1.3512g mannitol (molecular weight 180.16, target concentration 75mmol / L), 0.04g EDTA K2 (molecular weight 404.46, target concentration 1mmol / L), 0.12114g Tris (molecular weight 121.14, target concentration 10mmol / L, pH 7.2). Add these substances to an appropriate amount of deionized water in turn, stir evenly with a magnetic stirrer, and finally dilute to 100mL with deionized water.

[0064] Phosphate buffer (pH 7.4): Accurately weigh 1.36 g of potassium dihydrogen phosphate (molecular weight 136.09), place it in a beaker, slowly add 79 mL of 0.1 M sodium hydroxide solution (purchased from Shanghai Shenggong Company, product number A620617-0500), stir evenly with a magnetic stirrer, and then dilute to 200 mL with deionized water to obtain a phosphate buffer containing 50 mM potassium dihydrogen phosphate and 0.1 mol / L NaOH, pH 7.4.

[0065] Lysis buffer: Weigh 0.503 g ammonium chloride (molecular weight 53.49, target concentration 144 mmol / L), 0.404 g EDTA K2 (molecular weight 404.46, target concentration 1 mmol / L), and 0.084 g sodium bicarbonate (molecular weight 84.01, target concentration 1 mmol / L). Add these substances to an appropriate amount of deionized water, stir with a magnetic stirrer until completely dissolved, and then dilute to 1 L with deionized water. Use KOH solution to slowly adjust the pH to 8.0 to obtain a lysis buffer.

[0066] All prepared solutions were sealed and stored in a refrigerator at 4°C.

[0067] Example 1 Extraction of mitochondrial proteins

[0068] 1. Mitochondria Extraction

[0069] Use professional blood collection equipment to collect 3mL (3-5mL) of peripheral venous blood, and transfer the collected blood sample immediately to a centrifuge tube. Place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 2800rpm for 6 minutes. After the centrifugation is completed, carefully remove the centrifuge tube. At this time, you can see that the blood sample is clearly divided into two layers, the upper layer is plasma, and the lower layer is blood cells. Use a pipette to slowly absorb and discard the upper layer of plasma, and be careful to avoid aspirating the blood cell layer during the operation.

[0070] Transfer the blood cells in the lower layer to a 10mL centrifuge tube, and use a pipette to accurately add 4mL (4-6mL) of lysis buffer to make the total volume of the solution reach about 8mL. Gently invert the centrifuge tube to fully mix the blood cells and lysis buffer, and then let the centrifuge tube stand for 15 minutes, shaking it gently every 3 minutes to ensure that the red blood cells are fully lysed. Centrifuge the lysed solution again at 2800rpm for 6 minutes. After the centrifugation is completed, carefully discard the supernatant and retain the precipitate.

[0071] Add 8 mL of lysis buffer to the centrifuge tube with the pellet, and use a pipette to gently blow the pellet to resuspend it in the lysis buffer. Let the centrifuge tube stand for 9 minutes, shake it every 3 minutes, and then centrifuge it at 2800 rpm for 6 minutes, discard the supernatant, and keep the white blood cell pellet.

[0072] Next, add 8 mL of saline to the centrifuge tube containing the leukocyte pellet. Use a pipette to mix the pellet, then centrifuge at 2800 rpm for 6 minutes and discard the supernatant. Repeat this step once to further wash the pellet. Use a pipette to remove as much residual saline as possible.

[0073] Then add 2mL of leukocyte resuspension solution to the centrifuge tube containing the washed leukocyte pellet, and use a pipette to gently blow and mix the pellet to ensure that the leukocytes are completely resuspended. Carefully transfer the resuspension to a tightly fitting Dounce homogenizer, place the homogenizer on an ice bath, and use the matching pestle to slowly homogenize up and down 20 times to fully break the leukocytes. During the homogenization process, be careful to keep the homogenizer vertical to avoid uneven homogenization. Transfer the broken leukocyte solution to a 10mL centrifuge tube, rinse the homogenizer with 1mL of leukocyte resuspension solution, and transfer the rinse solution to the centrifuge tube to ensure that all broken leukocytes are collected.

[0074] Centrifuge the centrifuge tube containing the broken leukocyte solution at 2800rpm for 10 minutes. After the centrifugation, carefully collect the supernatant into two 1.5mL centrifuge tubes, being careful not to suck up the precipitate. Centrifuge the collected supernatant at 10600rpm for 10 minutes. After the centrifugation, discard the supernatant. The precipitate at the bottom of the tube is the mitochondria. Add 100μL of phosphate buffer to each centrifuge tube, use a pipette to gently blow the precipitate to resuspend the mitochondria, and then combine the mitochondrial solutions in the two centrifuge tubes.

[0075] 2. Acquisition of mitochondrial proteins

[0076] Prepare a 200 mL beaker, add ice cubes and fill it with water to form an ice bath. Insert the centrifuge tube containing the mitochondrial solution into the ice bath so that the bottom of the centrifuge tube touches the ice cubes, and let it cool for 3 minutes to ensure that the mitochondrial solution is ultrasonically disrupted in a low temperature environment to reduce the possibility of protein denaturation.

[0077] In an ice bath, slowly place the probe of the ultrasonic disruptor into the centrifuge tube, making sure that the probe is below the liquid surface but not touching the wall of the centrifuge tube. Turn on the ultrasonic disruptor, set the power to 50%, the working time to 1 min, the ultrasonic time to 2 s, and the interval time to 2 s, and use ultrasound to disrupt the mitochondria. During the ultrasonic process, closely observe the state of the solution to ensure that the ultrasound is carried out evenly. After the ultrasound is completed, immediately remove the centrifuge tube to obtain the mitochondrial protein solution. If it cannot be detected in time, it can be placed in a 4°C refrigerator for a short period of time for subsequent experiments. After the ultrasonic disruption is completed, use ultrapure water to carefully clean the ultrasonic disruptor probe to avoid residual substances affecting the next use.

[0078] 3. Detection of mitochondrial protein concentration

[0079] The mitochondrial protein concentration can be detected by a commonly used protein concentration detection method in a laboratory, such as the Bradford method, the BCA method, etc. In this embodiment, the mitochondrial protein concentration is detected by the Bradford method.

[0080] The mitochondrial protein solution stored in a 4°C refrigerator needs to be placed on a vortexer before testing, vortex mixed for 30 seconds to evenly distribute the protein in the solution, and then briefly centrifuged to remove any bubbles that may be generated.

[0081] According to the instructions of the Bradford protein concentration determination kit, prepare the standard bovine serum albumin (BSA) and G-250 staining solution. Dilute the standard in a gradient manner, mix the standard solutions of each concentration (0.05, 0.1, 0.2, 0.4, 0.8, 1 μg / μL) with the G-250 staining solution, and let it stand at room temperature for 3-5 minutes to fully react. After the reaction, use an enzyme reader to measure the absorbance value (OD value) at 595nm and draw a standard curve. Take an appropriate amount of mitochondrial protein solution and mix it with the G-250 staining solution, and let it stand at room temperature for 3-5 minutes to fully react. After the reaction, use an enzyme reader to measure the absorbance value (OD value) at 595nm. Calculate the protein concentration in the mitochondrial protein solution according to the standard curve.

[0082] 4. Results

[0083] The OD values ​​of the standard BSA with different concentrations are shown in Table 1 below. The standard curve is shown in Figure 1 As shown,

[0084] Table 1

[0085] Serial number BSA concentration (μg / μL) OD value 1 0.05 0.117 2 0.1 0.175 3 0.2 0.289 4 0.4 0.539 5 0.8 1.034 6 1 1.367

[0086] The protein concentration in the mitochondrial protein solution was calculated based on the standard curve to be 0.34 μg / μL.

[0087] Comparative Example 1

[0088] 1. Mitochondria Extraction

[0089] Collect 3 mL (2-3 mL) of peripheral venous blood, add 5 mL of lysis buffer, let stand for 9 minutes, shake every 3 minutes to lyse red blood cells, then centrifuge at 2800 rpm for 6 minutes and discard the supernatant.

[0090] Add 8 mL of lysis buffer to the centrifuge tube containing the precipitate, let stand for 9 minutes, shake every 3 minutes, centrifuge at 2800 rpm for 6 minutes, and discard the supernatant.

[0091] Then, add 8 mL of physiological saline to the centrifuge tube retaining the precipitate, blow and mix the precipitate, and centrifuge at 2800 rpm for 6 minutes. Repeat this step once, remove the supernatant, and obtain the leukocyte precipitate.

[0092] The obtained leukocytes were resuspended in 5 ml of leukocyte resuspension solution, mixed and homogenized 20 times using a tightly fitting Dounce homogenizer to break the leukocytes, followed by centrifugation at 2800 rpm for 10 minutes, and the supernatant was collected into a 1.5 mL centrifuge tube. The supernatant was centrifuged at 10600 rpm for 10 minutes to enrich the mitochondrial crude precipitate, and centrifuged again at 10600 rpm for 10 minutes to obtain a mitochondrial precipitate. The mitochondrial precipitate was resuspended in phosphate buffer to obtain a mitochondrial solution, which can be stored at -80°C.

[0093] 2. Acquisition of mitochondrial proteins

[0094] According to the method for obtaining mitochondrial protein in Example 1, the mitochondrial solution was disrupted by an ultrasonic disruptor to obtain a mitochondrial protein solution.

[0095] 3. Detection of mitochondrial protein concentration

[0096] According to the method for detecting mitochondrial protein concentration in Example 1, combined with the standard curve in Example 1, it was calculated that the protein concentration in the mitochondrial protein solution obtained by the method of this comparative example was 0.25 μg / μL.

[0097] When the same blood sample was extracted using the method of Example 1, the amount of mitochondrial protein extracted was increased by 36% compared to the method of this comparative example, and the extraction efficiency was significantly improved.

[0098] Comparative Example 2

[0099] The extraction method of mitochondrial protein in this comparative example is the same as that in Example 1, except that the lysis buffer in Example 1 is replaced with 0.1 mmol / L EDTA K2, pH 8.0.

[0100] The results showed that no leukocyte pellet could be obtained after centrifugation at 2800 rpm.

[0101] Comparative Example 3

[0102] The extraction method of mitochondrial protein in this comparative example is the same as that in Example 1, the only difference is that the mixing of blood cells with lysis buffer and standing for 15 minutes (the standing time after the first mixing with lysis buffer) in Example 1 is replaced by standing for 30 minutes after mixing.

[0103] The results showed that the protein concentration in the obtained mitochondrial protein solution was 0.27 μg / μL.

[0104] Comparative Example 4

[0105] The extraction method of mitochondrial protein in this comparative example is the same as that in Example 1, the only difference is that the ultrasonic conditions of the ultrasonic disruptor in Example 1 are adjusted to 90% power, 1 min working time, 10 s ultrasonic time, and 10 s rest time.

[0106] The results showed that the protein concentration of the obtained mitochondrial protein solution was 0.16 μg / μL. This may be due to the high power and long single ultrasonic time, which caused the solution in the centrifuge tube to splash out and the temperature to rise, leading to protein degradation.

[0107] Example 2 A device for extracting mitochondrial protein

[0108] 1. Device for extracting mitochondrial proteins

[0109] A device for extracting mitochondrial protein, the device is an integrated experimental chamber, the structural schematic diagram of the integrated experimental chamber is as shown Figure 2 and Figure 3 As shown, the integrated experimental cabin comprises an experimental system (1), a lighting system (2) and a ventilation system (3); the experimental system (1) of the integrated experimental cabin is as shown Figure 3 As shown, the experimental system (1) can provide a space and instruments for conducting experiments, the lighting system (2) can provide uniform and appropriate light, and the ventilation system (3) can adjust the air flow and humidity in the cabin to maintain a stable experimental environment; the experimental system (1) includes: a reagent preparation area (11), a sample processing area (12) and a detection area (13), and the areas are interconnected through a sealed channel (14) and an automatic isolation door (15); a spacer is set between the reagent preparation area (11) and the sample processing area (12), and between the sample processing area (12) and the detection area (13). The invention discloses a sample transfer window (16) and a high-efficiency air filtration system (17). The sample transfer window (16) is equipped with an automatic opening and closing device and a sample fixing clamp. The high-efficiency air filtration system (17) can effectively prevent air cross-contamination between various areas. A buffer room (18) is provided at the entrance of each of the reagent preparation area (11), the sample processing area (12) and the detection area (13). A biological safety cabinet (19) is also provided at the entrance of each of the reagent preparation area (11) and the sample processing area (12). The lighting system (2) and the ventilation system (3) are arranged on the top of the integrated experimental cabin.

[0110] The reagent preparation area (11) contains a variety of instruments and equipment for reagent preparation, including an intelligent reagent storage cabinet, a high-precision electronic balance, an automatic stirrer, an intelligent temperature control system, a disinfection device (ultraviolet lamp, etc.) and a laboratory table. The intelligent reagent storage cabinet has multiple layers of storage racks, each of which is equipped with an independent temperature and humidity sensor and an adjustment device, which can accurately control the storage environment of the reagents. The high-precision electronic balance can be accurate to 0.0001g and is used to accurately weigh the reagent components. The automatic stirrer can fully stir the reagent according to the preset stirring speed and time. The intelligent temperature control system can adjust the reagent to the required precise temperature and maintain the temperature stability.

[0111] The sample processing area (12) contains a variety of instruments and equipment for sample processing, including a multifunctional centrifuge, an intelligent vortexer, an automated ultrasonic disruptor, a special Dounce homogenizer and matching fixtures, disinfection equipment (UV lamp, etc.) and a laboratory table. The multifunctional centrifuge has a variety of centrifugal modes and adjustable centrifugal force. Its centrifuge tube slot can adapt to centrifuge tubes of different specifications, and an elastic buffer pad and a sample positioning device are arranged inside the slot to effectively prevent the centrifuge tube from breaking and the sample from shaking. The intelligent vortexer can vortex the sample according to the preset vortex speed and time, and has an automatic stop function. The automated ultrasonic disruptor has adjustable ultrasonic power, frequency and time. Its ultrasonic probe is positioned by a high-precision mechanical arm, which can accurately control the position and range of ultrasonic disruption. The surface of the ultrasonic probe is also coated with a special anti-corrosion and anti-pollution coating. The special Dounce homogenizer and matching fixture are made of special materials and designs, which can more effectively disrupt cells. The matching fixture has a clamping and loosening function and can be automatically adjusted according to the size of the homogenizer.

[0112] The detection area (13) contains a variety of instruments and equipment for detecting experimental results, including a high-precision protein concentration detection kit, an ELISA reader, and a data processing system. The high-precision protein concentration detection kit can quickly and accurately detect the concentration of mitochondrial proteins, and the data processing system can analyze, record, and store the detection data in real time, and can transmit and share data with external devices.

[0113] 2. Assembly and debugging of the device

[0114] (1) Install an integrated experimental cabin at a suitable location in the laboratory, connect the sealed passages (14) and automatic isolation doors (15) between the various areas according to the design requirements, ensure the sealing of the sealed passages (14) and the normal opening and closing functions of the automatic isolation doors (15), install the buffer rooms (18) and biological safety cabinets (19) at the entrances of the reagent preparation area (11), sample processing area (12) and detection area (13), install the sample transfer windows (16) and high-efficiency air filtration systems (17) between the various areas, and install the automatic opening and closing device and sample fixing fixture on the sample transfer window (16). Install the lighting system (2) and ventilation system (3) on the top of the experimental cabin, and debug them to ensure that the lighting is uniform and appropriate, and the ventilation system can stably adjust the air flow and humidity in the cabin.

[0115] (2) Install an intelligent reagent storage cabinet, a high-precision electronic balance, an automatic stirrer, an intelligent temperature control system, a disinfection device (UV lamp, etc.) and a laboratory bench in the reagent preparation area (11). Set up the intelligent reagent storage cabinet according to the instructions, calibrate the high-precision electronic balance, and debug the automatic stirrer and intelligent temperature control system to ensure their normal operation.

[0116] (3) Install a multifunctional centrifuge, an intelligent vortexer, an automated ultrasonic disruptor, a special Dounce homogenizer and matching fixtures, disinfection equipment (UV lamp, etc.) and a laboratory bench in the sample processing area (12). Debug the multifunctional centrifuge with different centrifugal modes and centrifugal forces, calibrate the vortex speed and time of the intelligent vortexer, debug the ultrasonic power, frequency and time of the automated ultrasonic disruptor, and check the clamping and loosening functions and size adjustment functions of the special Dounce homogenizer and matching fixtures.

[0117] (4) A protein concentration detection kit or a protein concentration detection device is provided in the detection area (13).

[0118] 3. Use of the device

[0119] After the device is assembled and debugged, the lighting system (2), ventilation system (3) and high-efficiency air filtration system (17) are turned on, and the experimenter enters the experimental cabin and then enters each area through the buffer room (18) at the entrance of the reagent preparation area (11), sample processing area (12) and detection area (13).

[0120] First, in the reagent preparation area (11), select, weigh, stir, and adjust the temperature of the reagents in strict accordance with the operating requirements of the reagent preparation stage. When selecting reagents in the intelligent reagent storage cabinet, pay attention to the validity period and storage conditions of the reagents to ensure that the reagents used meet the requirements. When using a high-precision electronic balance to weigh the reagents, place the balance in a stable position to avoid vibration that affects the weighing accuracy. When stirring the reagents, set the stirring speed and time reasonably according to the properties and requirements of the reagents to ensure that the reagents are fully stirred.

[0121] Before entering the experimental cabin, the experimenter should change into a special lab coat, make sure the cuffs are tied tightly, put on gloves on both hands (gloves must completely cover the wrists), and wear a protective mask. Turn on the ultraviolet light in the experimental cabin half an hour in advance for comprehensive disinfection. After disinfection is completed, use 75% alcohol to carefully wipe the surface of the test bench. Specifically, the experimenter enters the experimental cabin and first prepares the sample to be tested (collected peripheral blood sample), physiological saline, white blood cell resuspension, phosphate buffer (pH7.4), lysis buffer, standard bovine serum albumin gradient dilution solution and other reagents in the reagent preparation area (11).

[0122] Next, the sample to be tested and other reagents are transferred to the sample processing area (12) through the sample transfer window (16) between the reagent preparation area (11) and the sample processing area (12), the reagents are fixed by the sample fixing clamp, the automatic opening and closing device is opened, and the reagents are transferred to the sample processing area (12) through the sample transfer window (16). The sample to be tested is processed in the sample processing area (12), and the protein in the peripheral blood mitochondria is extracted to obtain a mitochondrial protein solution. During the sample processing stage, the operator needs to pay close attention to the processing of the sample in each instrument. During the centrifugation process, check whether the centrifuge tube is placed correctly and whether the centrifugal force is appropriate to avoid sample loss or contamination due to improper centrifugation. During the vortexing, ultrasonic disruption and homogenization operations, the operation is performed according to the preset parameters to ensure the accuracy and effectiveness of the operation.

[0123] When transferring samples, operate through the sample transfer window and be careful to keep the sample transfer window clean and closed to prevent the sample from being contaminated.

[0124] Specifically, sample processing includes the following steps:

[0125] S1. Centrifuge the peripheral blood sample at 2800 rpm for 6 minutes. After centrifugation, discard the upper plasma to obtain blood cells.

[0126] S2. Add lysis buffer to the obtained blood cells, mix well, and let stand for 15 minutes. During this period, gently shake once every 3 minutes to ensure that the red blood cells are fully lysed. Then centrifuge at a speed of 2800rpm for 6 minutes, discard the supernatant, add lysis buffer to the obtained precipitate, mix well and let stand for 9 minutes, shake once every 3 minutes, and then centrifuge at a speed of 2800rpm for 6 minutes, discard the supernatant, and obtain a white blood cell precipitate. The composition of the lysis buffer is: water, 144mmol / L ammonium chloride, 1mmol / LEDTA K2 and 1mmol / L sodium bicarbonate.

[0127] S3. The obtained leukocyte precipitate was washed and purified with physiological saline, and after washing, it was centrifuged at 2800 rpm for 6 minutes, and the supernatant was discarded to obtain a purified leukocyte precipitate.

[0128] S4. Add leukocyte resuspension to the purified leukocyte precipitate, mix well, and homogenize in an ice bath for 20 times to fully break the leukocytes. Fully collect the homogenized leukocyte homogenate (i.e., the solution containing broken leukocytes), centrifuge at 2800 rpm for 10 minutes, collect the supernatant, centrifuge the collected supernatant at 10600 rpm for 10 minutes, discard the supernatant, and the precipitate is the mitochondria. Add phosphate buffer to the precipitate, mix well, and obtain a mitochondrial solution. The composition of the leukocyte resuspension is: water, 0.1 g / L digitonin, 25 mmol / L sucrose, 75 mmol / L mannitol, 1 mmol / L EDTA K2, and 10 mmol / LTris (pH 7.2);

[0129] The composition of phosphate buffer is: water, 50 mM potassium dihydrogen phosphate and 0.1 mol / L NaOH.

[0130] S5. In an ice bath state, the mitochondrial solution is broken by an ultrasonic disruptor, the power is set to 50%, the working time is 1 min, the ultrasonic time is 2 s, the interval time is 2 s, and the mitochondria are broken by ultrasonic waves to obtain a mitochondrial protein solution, that is, mitochondrial protein.

[0131] Finally, the processed sample (mitochondrial protein solution) is transferred to the detection area (13) through the sample transfer window (16) between the sample processing area (12) and the detection area (13), the sample is fixed by a sample fixing fixture, the automatic opening and closing device is opened, and the sample is transferred to the detection area (13) through the sample transfer window (16). The sample is used in the detection area (13) to obtain detection data using a test kit and a detection device, and the data is analyzed to obtain a detection result.

[0132] During the testing phase, when the operator uses the kit to test the extracted mitochondrial protein solution, he / she should ensure that the kit solution is in good condition and the test data is accurate. After obtaining the test data, it is ensured that it is analyzed, recorded and stored in a timely manner, and the operator can view the relevant data as needed.

[0133] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting mitochondrial protein, characterized in that: The following steps are involved: S1, centrifuge peripheral blood, collect precipitate and obtain blood cells; S2. Add lysis buffer to the blood cells obtained in step S1, mix well, let stand, repeat the mixing and letting stand, the total standing time is 15-20 minutes; Then, centrifuge and collect the precipitate, add lysis buffer to the precipitate and mix, let stand, repeat the mixing and letting stand for a total of 9-10 minutes, centrifuge and collect the leukocyte precipitate; The lysis buffer comprises water, 140-150 mmol / L ammonium chloride, 0.8-1.2 mmol / L EDTA K2 and 0.8-1.2 mmol / L sodium bicarbonate, with a pH value of 8.0; S3, the leukocyte precipitate obtained in step S2 is washed and purified with physiological saline to obtain a purified leukocyte precipitate; S4. Add the leukocyte resuspension solution to the leukocyte precipitate purified in step S3, mix well, and homogenize in an ice bath; fully collect the homogenized leukocyte homogenate, centrifuge, collect the supernatant, centrifuge the supernatant, collect the precipitate, add phosphate buffer to the precipitate, mix well, and obtain a mitochondrial solution; The composition of the leukocyte resuspension solution is: water, 0.1-0.2 g / L digitonin, 20-25 mmol / L sucrose, 75-80 mmol / L mannitol, 1-1.5 mmol / L EDTA K2 and 10-12 mmol / L Tris; S5. In an ice bath state, the mitochondrial solution obtained in step S4 is disrupted by an ultrasonic disruptor to obtain a mitochondrial protein solution.

2. The method according to claim 1, characterized in that In steps S1 and S2, the centrifugation conditions are both 2500-3000 rpm for 5-8 minutes; And / or, in step S2, the repeated mixing and letting stand is repeated mixing and letting stand for 1-3 minutes.

3. The method according to claim 1, characterized in that Preferably, in step S3, the cleaning is performed at least twice; And / or, in step S4, the homogenization is performed at least 20 times; and / or, in step S4, the fully collecting includes collecting the leukocyte homogenate remaining in the homogenizer; and / or, in step S4, the leukocyte homogenate is centrifuged at 2500-3000 rpm for 10-15 minutes; And / or, in step S4, the supernatant is centrifuged at 10600-11000 rpm for 10-15 minutes; And / or, in step S4, the composition of the phosphate buffer is: water, 50-55 mM potassium dihydrogen phosphate and 0.1-0.2 mol / L NaOH.

4. The method according to claim 1, characterized in that: In step S5, the conditions for the ultrasonic crusher to crush are: power 50%, working time 1 min, ultrasonic time 2-4 s, and intermittent time 2-4 s.

5. A device for extracting mitochondrial protein using the method according to any one of claims 1 to 4, characterized in that: The device is an integrated experimental cabin, which includes an experimental system (1), a lighting system (2) and a ventilation system (3); the experimental system (1) can provide a space and instruments for conducting experiments, the lighting system (2) can provide uniform and appropriate light, and the ventilation system (3) can adjust the air flow and humidity in the cabin to maintain the stability of the experimental environment; the experimental system (1) includes: a reagent preparation area (11), a sample processing area (12) and a detection area (13), and the areas are connected to each other through a sealed channel (14) and an automatic isolation door (15); the reagent preparation area (11) A sample transfer window (16) and a high-efficiency air filtration system (17) are arranged between the sample processing area (12) and the detection area (13); the sample transfer window (16) is equipped with an automatic opening and closing device and a sample fixing clamp; the high-efficiency air filtration system (17) can effectively prevent air cross contamination between various areas; a buffer room (18) is arranged at the entrance of each of the reagent preparation area (11), the sample processing area (12) and the detection area (13); and a biological safety cabinet (19) is also arranged at the entrance of each of the reagent preparation area (11) and the sample processing area (12).

6. The device according to claim 5, characterized in that The lighting system (2) and the ventilation system (3) are arranged on the top of the integrated experimental cabin.

7. The device according to claim 5, characterized in that The reagent preparation area (11) is provided with instruments and equipment used for reagent preparation, including an intelligent reagent storage cabinet, a high-precision electronic balance, an automatic stirrer, an intelligent temperature control system, a disinfection device and a laboratory table.

8. The device according to claim 5, characterized in that The sample processing area (12) is provided with instruments and equipment for sample processing, including a multifunctional centrifuge, an intelligent vortex instrument, an automatic ultrasonic disruptor, a Dounce homogenizer and matching fixtures, disinfection equipment and a laboratory table.

9. The device according to claim 5, characterized in that The detection area (13) is provided with instruments and equipment for detecting the experimental results, including a high-precision protein concentration detection kit, an enzyme marker and a data processing system.

10. The device according to any one of claims 7 to 9, characterized in that: The intelligent reagent storage cabinet has multiple layers of storage racks, each of which is equipped with independent temperature and humidity sensors and adjustment devices, which can accurately control the storage environment of the reagents; the high-precision electronic balance can be accurate to 0.0001g, which is used to accurately weigh the reagent components; the automatic stirrer can fully stir the reagents according to the preset stirring speed and time; the intelligent temperature control system can adjust the reagents to the required precise temperature and maintain the temperature stability; And / or, the multifunctional centrifuge has multiple centrifugal modes and adjustable centrifugal force, its centrifuge tube slot can adapt to centrifuge tubes of different specifications, and an elastic buffer pad and a sample positioning device are arranged inside the slot, which can effectively prevent the centrifuge tube from breaking and the sample from shaking; the intelligent vortex instrument can vortex the sample according to the preset vortex speed and time, and has an automatic stop function; the automated ultrasonic disruptor has adjustable ultrasonic power, frequency and time, and its ultrasonic probe is positioned by a high-precision mechanical arm, which can accurately control the position and range of ultrasonic disruption, and the surface of the ultrasonic probe is also coated with a special anti-corrosion and anti-pollution coating; the Dounce homogenizer and the matching clamp are made of special materials and designs, which can more effectively disrupt cells, and the matching clamp has a clamping and loosening function, and can be automatically adjusted according to the size of the homogenizer; And / or, the high-precision protein concentration detection kit can quickly and accurately detect the concentration of mitochondrial proteins; the data processing system can analyze, record and store the detection data in real time, and can transmit and share data with external devices.

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