CN115364521B - Preparation method of size exclusion chromatography separation column for exosome separation - Google Patents
Preparation method of size exclusion chromatography separation column for exosome separation Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
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Abstract
The invention discloses a preparation method of a size exclusion chromatography separation column for separating exosomes, which comprises material preparation, material cleaning and gel filling, and the separation column can be used for separating exosomes in liquid samples such as cell culture fluid supernatant, blood plasma, urine and the like. The method reduces the time consumption of the separation process, reduces the pollution of high-abundance proteins and other impurities in the separation process, does not need complex instruments and equipment, and greatly reduces the cost.
Description
Technical Field
The invention relates to a preparation method of a size exclusion chromatography separation column for separating exosomes.
Technical Field
Exosomes (exosomes) are a membranous vesicle released outside the cell from the intracellular vesicle structure, with a diameter of about 40-160nm (average diameter 100 nm). Exosome-like vesicles are secreted into the extracellular space by exocytosis as a result of fusion between the plasma membrane and the multivesicular body. Exosome-like vesicles, which are divided into inner and outer by lipid bilayers, can contain many components of the cell, including DNA, RNA, lipids, metabolites, cytoplasm, and cell surface proteins. The main function of exosomes is to communicate intercellular information, typically through the transfer of biomolecules on the membrane surface or in the lumen, to regulate the normal physiological functions of the cells. In addition, the exosomes contain biomarkers of various diseases, and early diagnosis of diseases such as cancers, virus infection and the like is hopeful to be realized by utilizing the exosomes existing in a large amount in peripheral blood, so that the exosomes are research hot spots in the current academic world.
The existing separation methods for exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, size exclusion chromatography, polymer precipitation, affinity capture, and the like. Size exclusion chromatography (size exclusion chromatography, SEC) uses mesh openings in a spherical gel to allow molecules of different sizes to flow through the packing material through different paths, thereby eluting at different time periods: macromolecules cannot enter the gel sieve holes, run a shorter path and are eluted earlier; smaller molecules can enter the mesh holes in the gel, travel longer paths, and elute longer. The separation method can effectively separate the exosomes from the high-abundance proteins existing in the serum, and reduce the dependence on complex instruments and equipment.
Disclosure of Invention
The invention aims to provide a preparation method of a size exclusion chromatography separation column for exosome separation. The separation column can be used for separating exosomes in liquid samples such as cell culture fluid supernatant, blood plasma, urine and the like. The method can reduce the time consumption of the exosome separation process, reduce the pollution of high-abundance proteins and other impurities in the separation process, and greatly reduce the cost without complex instruments and equipment such as an ultra-high-speed centrifuge.
The invention aims at realizing the following scheme: a method for preparing a size exclusion chromatography separation column for exosome separation, comprising the steps of:
s1, preparing materials:
preparing needed tools including a separation column, an upper top cover, a lower opening plug, a sieve plate, a beaker, a pipettor, a disposable dropper, a filling column, a special pipe rack, gel for filling, absolute ethyl alcohol, deionized water and the like; preparing 75% ethanol solution for sterilization, and 20% ethanol solution as buffer solution;
s2, cleaning materials:
cleaning the separation column, the filling column, the upper top cover and the lower opening plug; placing in a clean large beaker, soaking in 75% ethanol solution for 10min for sterilization, then soaking in 20% ethanol solution, performing ultrasonic treatment for 20min, soaking for 10min, and drying at 37 ° for later use; the bagging is noted to avoid dust;
cleaning the sieve plate: soaking in 75% ethanol solution for 10min, sterilizing, soaking in 20% ethanol solution, and pulse ultrasonic treatment for 3 times each for 20min at intervals of 10min to empty bubbles. Soaking in 20% ethanol solution overnight, and performing ultrasonic treatment again for 20min before use; the bagging is noted to avoid dust;
s3, gel filling:
(1) A 20% ethanol solution infiltrates the separation column, the upper top cover and the lower opening plug;
(2) And (3) mounting a lower sieve plate: washing the inner wall of the column with 20% ethanol solution, and then mounting a sieve plate; slowly pushing the lower sieve plate to the bottom by using a filling column, taking care to avoid scratching the inner wall of the column; after the lower sieve plate is fixed, the upper and lower opening plugs are covered, and the filling column is slowly taken out; care must be taken slowly to prevent the generation of bubbles; filling 20% ethanol solution, removing the lower plug, and allowing ethanol to naturally flow out to remove bubbles generated in the process of filling the sieve plate; then the lower outlet was closed, leaving a small amount (0.5 cm) of ethanol in the column;
(3) Gel loading (8-10 cm): balancing to room temperature 2h before preparation, and filling at a speed of 0.5ml/min, wherein the solution can be drained by a glass rod or slowly dropped along the inner wall of the column by a dropper to avoid generating bubbles; ethanol is added at the top, and after filling is completed, the mixture naturally subsides to a stable state for at least 3 hours; the content of the filling gel can be adjusted during the process, and the gel is required to be stabilized again after the gel is changed;
(4) Opening the lower outlet, discharging the ethanol solution in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) Slowly adding 20% ethanol solution to fill the upper space, and opening the lower outlet; note that the gel must be slow, the steady state of the gel must not be broken, until the buffer (20% ethanol solution) can flow out steadily, i.e. the system is steady state, and then the lower outlet is closed;
(6) In the same way, a sieve plate is arranged and is slowly pushed in;
(7) Opening a lower outlet, naturally settling, and slightly pressing down the sieve plate by 2-3mm;
(8) Closing the lower outlet, naturally settling, adding 20% ethanol solution if the upper part has a space, reserving a space of 0.5cm, and sealing the top cover without filling up;
(9) Marking, and storing in a refrigerator at 4 ℃.
The gel filler adopted by the size exclusion chromatography in the step S1 is Sepharose CL-2B, and the characteristics are as follows: the particle diameter is 60-200 μm, and the separation range of the globular protein is 70,000-40X 106 kDa.
The gel filler adopted by the size exclusion chromatography in the step S1 is Sepharose CL-4B, and the characteristics are as follows: the particle diameter is 45-165 μm, and the separation range of the globular protein is 60,000-20X 106 kDa.
The gel filler adopted by the size exclusion chromatography in the step S1 is Sepharose CL-6B, and the characteristics are as follows: the particle diameter is 44-165 μm, and the separation range of the globular protein is 10,000-4X 106 kDa.
And (2) the bed volume of the size exclusion chromatography column in the step S1 is 10-12 mL.
The size exclusion chromatography described in step S2 uses a 20% ethanol solution as buffer.
The buffer solution was filtered through a 0.22 μm filter.
The separation column can be used for separating exosomes in liquid samples such as cell culture fluid supernatant, blood plasma, urine and the like. The method of the invention can reduce the time consumption of the separation process, reduce the pollution of high abundance proteins and other impurities in the separation process, and does not need complex instruments and equipment, thereby greatly reducing the cost. The method of the invention has the following advantages:
1. the raw material cost is low, and the preparation process is relatively simple;
the SEC separation column provided by the invention can be used for rapidly separating exosomes from a sample, does not need special equipment such as a super-high-speed centrifuge and the like, and is simple in separation operation;
the SEC separation column provided by the invention can be used for separating to obtain high-purity exosomes on the premise of keeping the whole morphology and function of exosomes;
the SEC separation column provided by the invention can remove free protein in a sample and has strong bearing capacity on the free protein;
and 5. The SEC separation column provided by the invention does not introduce impurities from non-sample sources in the separation process.
Drawings
The SEC separation column prepared in fig. 1;
the reference numerals in the figures illustrate:
1-a separation column; 11—gel filled region;
2-upper top cover; 3-lower plug; 4. 5-upper and lower screen plates.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The starting materials used in the examples were all commercially available from conventional sources unless otherwise specified.
Example 1
The SEC separation column, as shown in figure 1, comprises a separation column 1, an upper top cover 2 and a lower port plug 3, wherein the separation column 1 is provided with a gel filling area 11, and an upper sieve plate 4 and a lower sieve plate 5 are arranged above and below the gel filling area 11, and the SEC separation column is prepared according to the following steps:
s1, preparation of materials
Instrument preparation: comprises a separation column 1, an upper top cover 2, a lower opening plug 3, an upper sieve plate 4 and a lower sieve plate 5;
an operation tool: beaker, pipettor, disposable dropper, filling column and/or special tube rack;
material preparation: gel for filling CL-2B, absolute ethyl alcohol, deionized water and the like, and preparing 75% ethanol solution for sterilization; a 20% ethanol solution used as a buffer;
s2, cleaning the material
Cleaning a separation column 1, a filling column, an upper top cover 2 and a lower opening plug 3, placing the separation column, the filling column, the upper top cover 2 and the lower opening plug 3 in a clean large beaker, soaking the separation column in 75% ethanol solution for 10min, and sterilizing; then soaking in a buffer solution of 20% ethanol solution, carrying out ultrasonic treatment for 20min, soaking for 10min, drying at 37 ℃ for standby, and taking care of bagging to avoid dust;
cleaning the sieve plate: soaking in 75% ethanol solution for 10min for sterilizing; soaking in 20% ethanol solution of buffer solution, and pulse ultrasonic treatment for 3 times for 20min each time at intervals of 10min for evacuating air bubbles. Soaking in 20% ethanol solution overnight, and ultrasonic treating for 20min again before use, taking care of bagging to avoid dust;
s3, gel filling
(1) Soaking the separation column 1, the upper top cover 2 and the lower opening plug 3 by using a buffer solution 20% ethanol solution;
(2) And (5) mounting a lower sieve plate 5: washing the inner wall of the separation column 1 with 20% ethanol solution of buffer solution, then mounting a lower sieve plate 5, slowly pushing the lower sieve plate 5 into the bottom of the separation column 1 by using a filling column, avoiding scratching the inner wall of the separation column 1, after the lower sieve plate 5 is fixed, covering a top cover 2 and a lower opening plug 3, taking out the filling column (taking care of being slow, preventing generation of bubbles, and ensuring that the buffer solution is fully filled with 20% ethanol solution), taking out the lower opening plug 3, allowing the buffer solution to naturally flow out, draining bubbles generated in the process of mounting the lower sieve plate 5, then closing a lower outlet, and leaving a small amount of buffer solution of 0.5cm in the separation column 1;
(3) Filling gel: gel Sepharose CL-2B is filled to the depth of 8-10cm, the gel Sepharose CL-2B is balanced to room temperature before being prepared, the filling speed is about 0.5ml/min, and the gel Sepharose CL-2B can be used for preventing bubbles and draining by a glass rod or can be slowly dripped along the inner wall of a separation column 1 by using a dropper; filling ethanol at the top, naturally settling for at least 3 hours until the gel column is in a stable state after filling, and stabilizing again after changing if the content of the filled gel is adjusted during the period;
(4) Opening the lower outlet, discharging the ethanol in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) Slowly adding a buffer solution of 20% ethanol solution to fill the upper space, opening the lower outlet, preventing breaking the stable state of gel until the buffer solution can stably flow out, namely, the system is in a stable state, and then closing the lower outlet;
(6) Installing a screen plate 4: slowly pushing the upper screen plate 4 in the same manner as the lower screen plate 5;
(7) Opening a lower outlet, and slightly pressing down the sieve plate for 2-3mm after the system naturally subsides;
(8) Closing the lower outlet, after the system naturally subsides, if the upper part of the separation column 1 has a space, adding buffer solution to a space with the height of 0.5cm, and sealing by using an upper top cover;
(9) Marking, and storing in a refrigerator at 4 ℃ for standby.
In this example, the gel packing used for size exclusion chromatography was Sepharose CL-2B, which has the following properties: the particle diameter is 60-200 μm, and the separation range of the spherical protein is 70,000-40×10 6 kDa。
The bed volume of the size exclusion chromatographic column is 10-12 mL.
Example 2
SEC separation columns, identical in structure to example 1, were prepared as follows:
s1, preparation of materials
Instrument preparation: comprises a separation column 1, an upper top cover 2, a lower opening plug 3, an upper sieve plate 4 and a lower sieve plate 5;
preparing a tool: beaker, pipettor, disposable dropper, filling column and/or special tube rack;
material preparation: gel for filling CL-4B, absolute ethyl alcohol and deionized water, and preparing 75% ethanol solution of disinfectant for sterilization; buffer 20% ethanol solution;
s2, cleaning the material
Cleaning a separation column 1, an upper top cover 2, a lower opening plug 3 and an operation tool filling column, placing the separation column in a clean large beaker, and soaking and sterilizing the separation column in 75% ethanol solution for 10min; then soaking in 20% ethanol solution of buffer solution, ultrasonic treating for 20min, soaking for 10min, oven drying at 37deg.C, and keeping dust (such as bagging to avoid dust);
cleaning the sieve plate: soaking in 75% ethanol solution for sterilization for 10min, soaking in 20% ethanol solution of buffer solution, performing pulsed ultrasound for 3 times for 20min each time at intervals of 10min, evacuating bubbles, soaking in 20% ethanol solution of buffer solution overnight, performing ultrasound for 20min again before use, and bagging for dust prevention;
s3, gel filling
(1) Soaking the separation column 1, the upper top cover 2 and the lower opening plug 3 by using a buffer solution 20% ethanol solution;
(2) And (5) mounting a lower sieve plate 5: after the inner wall of the separation column 1 is washed by buffer solution, the lower sieve plate 5 is arranged, the lower sieve plate 5 is slowly pushed into the bottom of the separation column 1 by a filling column, the inner wall is prevented from being scratched, after the lower sieve plate 5 is fixed, the upper and lower opening plugs 3 are covered, the filling column is slowly taken out to fill with 20% ethanol solution of the buffer solution, the lower opening plugs 3 are taken down, the buffer solution naturally flows out, so that bubbles generated in the process of filling the lower sieve plate 5 are completely discharged, then, the lower outlet is closed, and the buffer solution with the height of about 0.5cm is reserved in the separation column 1;
(3) Filling gel: gel Sepharose CL-4B loading 8-10cm; balancing to room temperature for 2 hours before preparation, filling at a filling speed of 0.5ml/min, draining with a glass rod or slowly dripping with a dropper along the inner wall of the separation column 1 to avoid generating bubbles, filling ethanol at the top of the separation column, naturally settling for at least 3 hours after filling, and stabilizing again after changing if the content of filled gel is adjusted;
(4) Opening the lower outlet, discharging the ethanol solution in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) The upper space was filled with a 20% ethanol solution slowly and the lower outlet was opened. (note that it must be slow to break the steady state of the gel) to a buffer (20% ethanol solution) that can flow steadily out, i.e. the system is steady state, then the lower outlet is closed.
(6) Installing a screen plate 4: slowly pushing the upper screen plate 4 in the same manner as the lower screen plate 5;
(7) Opening a lower outlet, naturally settling the system, and slightly pressing down the sieve plate by 2-3mm;
(8) Closing the lower outlet, naturally settling the system, if the upper part has a space, adding 20% ethanol solution of buffer solution, reserving a space with the height of 0.5cm, and sealing the upper top cover 2;
(9) Marking, and storing in a refrigerator at 4 ℃ for standby.
The gel filler adopted by the size exclusion chromatography in the embodiment is Sepharose CL-4B, and the characteristics are as follows: the particle diameter is 45-165 μm, and the separation range of the spherical protein is 60,000-20×10 6 kDa。
Example 3
Preparation of SEC separation column
S1, preparation of materials
Instrument preparation: comprises a separation column 1, an upper top cover 2, a lower opening plug 3, an upper sieve plate 4 and a lower sieve plate 5;
preparing a tool: beaker, pipettor, disposable dropper, filling column and/or special tube rack;
material preparation: gel for filling CL-6B, absolute ethyl alcohol and deionized water, and preparing 75% ethanol solution of disinfectant for sterilization; buffer 20% ethanol solution;
s2, cleaning the material
Cleaning a separation column 1, an upper top cover 2 and a lower opening plug 3, and operating a tool filling column, placing the separation column in a clean large beaker, soaking and sterilizing the separation column in a 75% ethanol solution for 10min, soaking the separation column in a buffer solution 20% ethanol solution, performing ultrasonic treatment for 20min, soaking for 10min, drying the separation column at 37 ℃, and bagging for ash prevention;
cleaning the sieve plate: sterilizing by soaking in 75% ethanol solution for 10min, soaking in buffer solution 20% ethanol solution, pulsed ultrasonic for 3 times each for 20min at intervals of 10min to empty bubbles, soaking in 20% ethanol solution overnight, and ultrasonic again for 20min before use, taking care of bagging to avoid dust.
S3, gel filling
(1) Buffer solution 20% ethanol solution infiltrates the separation column 1, the upper top cover 2 and the lower opening plug 3;
(2) And (5) mounting a lower sieve plate 5: washing the inner wall of the separation column 1 with 20% ethanol solution, and then mounting a sieve plate 5; the lower sieve plate 5 is slowly pushed into the bottom of the separation column 1 by a filling column, so that the inner wall of the separation column 1 is prevented from being scratched; after the lower sieve plate 5 is fixed, the upper and lower opening plugs 3 are covered, a filling column is used for slowly filling 20% ethanol solution of the buffer solution, the lower opening plugs 3 are taken down, the buffer solution naturally flows out to exhaust bubbles generated in the process of filling the lower sieve plate 5, then the lower outlet is closed, and a small amount (0.5 cm) of ethanol solution is left in the separation column 1;
(3) Filling gel: filling gel Sepharose CL-6B to 8-10cm, balancing to room temperature 2 hours before preparation, filling at 0.5ml/min, draining with a glass rod, or slowly dripping with a dropper along the inner wall of the separation column 1 to avoid generating bubbles, filling ethanol on the top, naturally settling for at least 3 hours after filling, and adjusting the content of filled gel until the system is in a stable state, wherein the gel is required to be stabilized again after the gel is changed;
(4) Opening the lower outlet, discharging the ethanol solution in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) Slowly adding a buffer solution, namely 20% ethanol solution, to fill the upper space of the separation column 1, opening the lower outlet until the buffer solution can flow out stably, namely closing the lower outlet after the system is in a stable state;
(6) Installing a screen plate 4: slowly pushing the upper screen plate 4 in the same manner as the lower screen plate 5;
(7) Opening the lower outlet, naturally settling the system, slightly pressing down the screen plate for 2-3mm
(8) Closing the lower outlet, naturally settling the system, if the upper part has a space, adding 20% ethanol solution of buffer solution, reserving a space of 0.5cm, and sealing the upper top cover 2;
(9) Marking, and storing in a refrigerator at 4 ℃ for standby.
In this example, the gel packing used for size exclusion chromatography was Sepharose CL-6B, which has the following properties: the particle diameter is 44-165 μm, and the separation range of the spherical protein is 10,000-4×10 6 kDa。
Claims (6)
1. A preparation method of a size exclusion chromatography separation column for exosome separation is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing materials: comprising
Preparing a separation column, an upper top cover, a lower opening plug, a sieve plate, a pipettor, a disposable dropper, a filling column and a special pipe rack;
filling gel, absolute ethyl alcohol and deionized water, preparing 75% ethanol solution for sterilization, and 20% ethanol solution for buffer solution;
s2, cleaning materials:
cleaning the separation column, the filling column, the upper top cover and the lower opening plug; placing the mixture in a clean large beaker, soaking in 75% ethanol solution for 10min for sterilization, soaking in 20% ethanol solution for 20min by ultrasound, soaking for 10min, and drying at 37 ℃ for later use;
cleaning the sieve plate: soaking in 75% ethanol solution for 10min, sterilizing, soaking in 20% ethanol solution, pulsed ultrasonic for 3 times, each time for 20min, and spacing for 10min to empty bubbles; then soaking the mixture in 20% ethanol solution overnight, and carrying out ultrasonic treatment again for 20min before use;
s3, gel filling:
(1) A 20% ethanol solution infiltrates the separation column, the upper top cover and the lower opening plug;
(2) And (3) mounting a lower sieve plate: washing the inner wall of the column with 20% ethanol solution, and then mounting a sieve plate; slowly pushing the lower sieve plate to the bottom by using a filling column, taking care to avoid scratching the inner wall of the column; after the lower sieve plate is fixed, the upper and lower opening plugs are covered, and the filling column is slowly taken out; filling 20% ethanol solution, removing the lower plug, and allowing ethanol to naturally flow out to exhaust bubbles generated in the process of filling the sieve plate; then closing the lower outlet, and reserving ethanol with the height of 0.5cm in the column;
(3) Filling gel: balancing to room temperature 2h before preparation, filling at a speed of 0.5ml/min, draining with a glass rod, or slowly dripping to 8-10cm along the inner wall of the column by using a dropper to avoid generating bubbles; adding ethanol at the top, standing for at least 3h after filling, and naturally settling to a stable state; if the gel filling amount needs to be adjusted, the gel needs to be kept still again for stabilization after the gel filling amount is changed;
(4) Opening the lower outlet, discharging the ethanol solution in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) Slowly adding 20% ethanol solution to fill the upper space, opening the lower outlet, and not breaking the stable state of gel until the buffer solution can flow out stably, i.e. closing the lower outlet after the system reaches the stable state;
(6) In the same way, a sieve plate is arranged and is slowly pushed in;
(7) Opening a lower outlet, naturally settling, and slightly pressing down the sieve plate by 2-3mm;
(8) Closing the lower outlet, naturally settling, adding 20% ethanol solution if the upper part has space, reserving 0.5cm space, and sealing the top cover;
(9) Marking, and storing in a refrigerator at 4 ℃.
2. The method according to claim 1, wherein the gel filler used in the size exclusion chromatography in step S1 is Sepharose CL-2B, which has the following properties: the particle diameter is 60-200 μm, and the separation range of the spherical protein is 70,000-40×10 6 kDa。
3. The method according to claim 1, wherein the gel filler used in the size exclusion chromatography in step S1 is Sepharose CL-4B, which has the following properties: the particle diameter is 45-165 μm, and the separation range of the spherical protein is 60,000-20×10 6 kDa。
4. The method according to claim 1, wherein the gel filler used in the size exclusion chromatography in step S1 is Sepharose CL-6B, which has the following properties: the particle diameter is 44-165 μm, and the separation range of the spherical protein is 10,000-4×10 6 kDa。
5. The method according to claim 1, wherein the size exclusion chromatography column of step S1 has a bed volume of 10-12 mL.
6. The preparation method according to any one of claims 1 to 2, characterized by comprising the steps of:
s1, instrument preparation: comprises a separation column (1), an upper top cover (2), a lower opening plug (3) and upper and lower sieve plates (4, 5);
an operation tool: beaker, pipettor, disposable dropper, filling column and/or special tube rack;
material preparation: gel for filling, namely Sepharose CL-2B, absolute ethyl alcohol, deionized water, and preparing 75% ethanol solution for sterilization; a 20% ethanol solution used as a buffer;
s2, cleaning the material
Cleaning a separation column (1), a filling column, an upper top cover (2) and a lower opening plug (3), placing the separation column, the filling column, the upper top cover (2) and the lower opening plug in a clean large beaker, soaking the separation column in 75% ethanol solution for 10min, and sterilizing; then soaking in a buffer solution of 20% ethanol solution, carrying out ultrasonic treatment for 20min, soaking for 10min, drying at 37 ℃ and dustproof for later use;
cleaning the sieve plate: soaking in 75% ethanol solution for 10min for sterilizing; soaking in 20% ethanol solution of buffer solution, and pulse ultrasonic treatment for 3 times for 20min each time at intervals of 10min for evacuating bubbles; soaking in 20% ethanol solution overnight, and performing ultrasonic treatment for 20min again before use, and bagging to prevent dust;
s3, gel filling
The method comprises the steps of (1) infiltrating a separation column (1), an upper top cover (2) and a lower opening plug (3) with a buffer solution of 20% ethanol;
(2) And (5) installing a lower sieve plate: washing the inner wall of the separation column (1) by using 20% ethanol solution of buffer solution, then mounting a lower sieve plate (5), slowly pushing the lower sieve plate (5) into the bottom of the separation column (1) by using a filling column, avoiding scratching the inner wall of the separation column (1), covering an upper top cover (2) and a lower opening plug (3) after the lower sieve plate (5) is fixed, taking out the filling column to fill up the buffer solution with 20% ethanol solution, taking out the lower opening plug (3), allowing the buffer solution to naturally flow out, draining bubbles generated in the process of mounting the lower sieve plate (5), then closing a lower outlet, and reserving 0.5cm of buffer solution in the separation column (1);
(3) Filling gel: gel Sepharose CL-2B is filled to the depth of 8-10cm, the gel Sepharose CL-2B is balanced to room temperature before being prepared, the filling speed is 0.5ml/min, and the gel Sepharose CL-2B is used for preventing bubbles, is drained by a glass rod, or is slowly dripped along the inner wall of a separation column 1 by using a dropper; filling ethanol at the top, naturally settling for at least 3 hours until the gel column is in a stable state after filling, and stabilizing again after changing if the content of the filled gel is adjusted during the period;
(4) Opening the lower outlet, discharging the ethanol in the column naturally, and closing the lower outlet after the gel is stabilized again;
(5) Slowly adding a buffer solution of 20% ethanol solution to fill the upper space, opening the lower outlet, preventing breaking the stable state of gel until the buffer solution can stably flow out, namely, the system is in a stable state, and then closing the lower outlet;
(6) Installing a screen plate (4): slowly pushing the upper screen plate (4) in the same manner as the lower screen plate (5);
(7) Opening a lower outlet, and slightly pressing down the sieve plate for 2-3mm after the system naturally subsides;
(8) Closing the lower outlet, after the system naturally subsides, if the upper part of the separation column (1) has a space, adding buffer solution to a space with the height of 0.5cm, and sealing by using an upper top cover;
(9) Marking, and storing in a refrigerator at 4 ℃ for standby.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20180126365A (en) * | 2017-05-17 | 2018-11-27 | 한국과학기술원 | Method for Isolation of Exosomes and Lipoproteins from Biological Sample |
| CN111961637A (en) * | 2020-07-08 | 2020-11-20 | 暨南大学 | Extracellular vesicle separation method based on combination of size exclusion chromatography and ultrafiltration |
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| US20100140173A1 (en) * | 2004-12-03 | 2010-06-10 | Chris Suh | Method and Device for Gravity Flow Chromatography |
| CN116322975A (en) * | 2020-09-16 | 2023-06-23 | 沃特世科技公司 | Adsorbents for improving chromatographic separation in size exclusion chromatography by reducing secondary interactions |
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| KR20180126365A (en) * | 2017-05-17 | 2018-11-27 | 한국과학기술원 | Method for Isolation of Exosomes and Lipoproteins from Biological Sample |
| CN111961637A (en) * | 2020-07-08 | 2020-11-20 | 暨南大学 | Extracellular vesicle separation method based on combination of size exclusion chromatography and ultrafiltration |
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