CN114292805A - Method for fully extracting total protein of adherent cells - Google Patents
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Abstract
The invention relates to a method for fully extracting total protein of adherent cells, which comprises the step of carrying out ultrasonic lysis on the adherent cells, wherein the ultrasonic lysis time is 2 min 30 s-3 min 30 s, the power is 30W-60W, the frequency is 15KHz-30KHz, the amplitude is 25-40 mu m, and the ultrasonic is switched on for 1-5 s and switched off for 4-8 s, so that the ultrasonic is circulated. According to the invention, by carrying out ultrasonic disruption when cells are in an adherent state, the destructive effect of shock waves and shearing force in liquid on the cells is increased, the ultrasonic disruption time is reduced, and the heat generated by the action of ultrasonic on a cell solution is reduced, so that the solution temperature is reduced, and the effects of improving the protein overflow efficiency and reducing the protein degradation are finally achieved. The method can more fully extract the total protein of the adherent cells, improve the measurement precision of the protein content of the adherent cells, save the step of digesting the cells and integrally shorten the cell lysis time.
Description
Technical Field
The invention relates to the field of cell biology, in particular to a method for fully extracting total protein of adherent cells.
Background
Currently, the measurement of total cellular protein content is mainly performed by BCA method and Coomassie Brilliant blue method. The BCA method has the characteristics of high accuracy and high detection speed. BCA (bicinchoninic acid) is mixed with a reagent consisting of other reagents such as cupric sulfate with bivalent copper ions to generate a green BCA working reagent. Protein Cu when BCA is combined with protein under alkaline condition2+Reduction to Cu+The working reagent changes from the original green color to a purple complex (see Smith, P.K., et al (1985) Measurement of protein using biochemical acid. anal Biochem150: 76-85). The light absorption intensity at 562nm is proportional to the protein concentration.
Regardless of which method is used, efficient cell disruption is a necessary prerequisite for accurate measurement of protein content. Currently, mechanical disruption, chemical and biochemical infiltration, and physical infiltration are used for cell disruption. The ultrasonic disruption belongs to mechanical disruption, and means that liquid generates cavitation under the action of ultrasonic waves, and a cavity is formed, enlarged and closed to generate great shock waves and shearing force so as to damage or disrupt a cell structure. The strength of the injury is closely related to the frequency and power of the ultrasound. At present, ultrasonic cell disruption is commonly used for extracting cell protein in a laboratory, but the effect of extracting protein by ultrasonic cell disruption is not good enough, and in order to overcome the problem that the harvested target protein is few and cannot be detected due to insufficient ultrasound, technicians in the field often need to enrich a large amount of cells, and enrich cells in a plurality of culture dishes together for suspension ultrasound, so that the purpose of enriching protein is finally achieved, and the requirement of fully releasing the protein is reduced. However, in the case of a small cell sample, such as the case of studying the content of a certain protein in cells obtained from a human body, it is critical to sufficiently release the protein from the limited cells if the content of the certain protein in a unit number of cells is to be accurately evaluated.
The degree of cell disruption when extracting the total cellular protein directly determines whether the total cellular protein content we have determined is accurate. After cell lysis due to ultrasonication, total intracellular protein will spill out of the cell. Even if the cell is disrupted, insufficient disruption can result in intra-packet retention of protein, resulting in inaccurate total protein measurements. On the other hand, increasing the time for disruption increases the cell disruption efficiency, but at the same time, generates a large amount of heat, thereby increasing the temperature of the protein solvent. It has been shown in the literature that higher temperatures degrade a portion of the protein. The long crushing operation affects the accuracy of the protein content measurement. Therefore, in order to accurately measure the total protein content, a method for efficiently disrupting cells while maintaining a short disruption time is required.
However, the method of cell suspension ultrasound used in the prior art has the following problems: first, because a large amount of cell sample is often required, it is not suitable for use in situations where the sample size is minimal or difficult to obtain; secondly, the situation of overhigh temperature can easily occur during the ultrasonic treatment, so that the protein is denatured and the target protein is lost; thirdly, because suspension ultrasound uses a large amount of cell samples, excessive temperature is avoided as much as possible, which in turn may lead to insufficient ultrasound and insufficient protein extraction.
Therefore, how to improve the cell ultrasonic efficiency is particularly important, and especially under the condition that the cell sample amount is small, the protein in the cells can be released more sufficiently, so that the accurate measurement of the protein content in the unit cell number becomes a difficult problem.
Disclosure of Invention
To the incomplete broken or problem that the protein degradation that higher temperature induced when broken of adherent cell can lead to the protein content survey inaccurate, the inventor is through carrying out the ultrasonication when the cell is adherent state, optimize the ultrasonic parameter, reduced the broken time of ultrasonic wave, reduce the supersound and to the cell solution effect and the heat that produces, thereby reduced the solution temperature, reach the effect that improves protein and spill over efficiency and reduce protein degradation, the final more abundant total protein of drawing adherent cell, the measurement accuracy of adherent cell protein content has been improved. In addition, compared with the ultrasonic treatment in a cell suspension state, the method also omits the step of digesting the cells, and shortens the time of cell lysis on the whole. Cells suspended by ultrasound are prone to foam, causing protein loss. And the ultrasonic adherent cells are not easy to generate foam in the solution, so that the loss of protein is further reduced.
Specifically, the present invention provides the following aspects:
in one aspect, the present invention provides a method for extracting total protein from adherent cells, comprising performing ultrasonic lysis on the cells in their adherent state, wherein the conditions of the ultrasonic lysis are as follows:
for a time of from 2 minutes 30 seconds to 3 minutes 30 seconds, preferably 2 minutes 40 seconds, 2 minutes 50 seconds, 3 minutes 10 seconds or 3 minutes 20 seconds,
the power is 30W-60W, including but not limited to 35W, 40W, 45W, 50W or 55W, preferably 45W,
the frequency is 15KHz-30KHz, including but not limited to 18KHz, 19KHz, 20KHz, 21KHz, 22KHz, 23KHz, 24KHz, 25KHz or 28KHz, preferably 21KHz,
an amplitude of 25-40 μm, including but not limited to 27 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 37 μm or 39 μm, preferably 35 μm,
ultrasound is switched on for 1-5 seconds and off for 4-8 seconds, and ultrasound is cycled, including but not limited to ultrasound being switched on for 2 seconds and off for 5 seconds, ultrasound being switched on for 3 seconds and off for 6 seconds, or ultrasound being switched on for 4 seconds and off for 7 seconds, preferably ultrasound being switched on for 2 seconds and off for 5 seconds.
In one embodiment, the method comprises adherent culture and sonication of adherent cells in a culture vessel having a diameter of less than 100mm, preferably less than 60 mm.
In one embodiment, the cell buffer used in the ultrasonic lysis is a protein-free cell buffer.
In one embodiment, the protein-free cell buffer is PBS.
In one embodiment, the height of the surface of the cell buffer used in the culture dish at the time of the ultrasonication is more than 1 cm.
In one embodiment, the sonication is performed under ice bath conditions.
In one embodiment, the cell is a cell capable of adherent growth, including but not limited to a fibroblast-type cell, such as a fibroblast, a cardiomyocyte, a smooth muscle cell, an osteoblast, or a vascular endothelial cell, an epithelial cell-type cell, such as a skin epithelial cell, an alimentary tract epithelial cell, a mammary epithelial cell, an alveolar epithelial cell, an epithelial tumor cell (e.g., a melanoma cell, an ovarian cancer cell), a migratory cell-type cell, such as a highly differentiated nasopharyngeal cancer cell, a polymorphous cell-type cell, such as a neural cell (e.g., a neuronal cell).
In one embodiment, the cells are sonicated at a cell density of 50% to 100% of the confluent culture dish, including but not limited to 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.
In one embodiment, the method does not include a stirring step upon sonication.
In another aspect, the present invention provides a method for accurately determining total protein of adherent cells, comprising extracting total protein of adherent cells according to the method for extracting total protein of adherent cells as described above, and determining the concentration of total protein using the BCA assay.
The "cell buffer" as used herein is the liquid used to soak the adherent cells during sonication. In some embodiments, the cell buffer is PBS.
Drawings
FIG. 1 shows the total protein concentration of MC3T3 osteoblasts (5X 10)5Individual cells, 13ml PBS);
FIG. 2 shows the total protein content of MC3T3 osteoblasts (5X 10)5Individual cells);
FIG. 3 shows the total protein concentration (5X 10) of A375 melanoma cells5Individual cells, 13ml PBS);
FIG. 4 shows the total protein content (5X 10) of A375 melanoma cells5Individual cells).
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments. The following examples are for the purpose of illustrating the invention only and should not be construed as limiting the invention in any way. The scope of protection of the invention is defined by the appended claims.
The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.
Example 1 investigation of cell disruption time
MC3T3 osteoblasts (purchased from GE), A375 melanoma cells (purchased from GE) at 5X10 per well5The cell number of (2) was injected into 6-well plates (diameter: 34.8mm) at 37 ℃ with 5% CO2Culture was performed under conditions with HyClone medium (purchased from GE) containing fetal bovine serum. After 24 hours, the cells were plated 80% and 100% onto the plates, and the cells in the 6-well plate were washed with 2ml PBS, and the PBS was discarded. Adding 200 mu L of pancreatin into 3 wells of a 6-well plate to digest the cells, stopping digestion by using the serum-containing culture medium and blowing off the cells into single cells after 3 minutes, washing the cells for three times by using PBS, and re-suspending the cells by 13mL of PBS and putting the cells back into the respective wells; to 3 additional wells of the 6-well plate, 13ml PBS was added per well (the level of cell buffer needed to exceed 1 cm). Adherent cells without trypsinization and suspended cells resuspended in PBS after trypsinization were subjected to ice-bath disruption using a cell disrupter (power 45W, frequency 21KHz, amplitude 35 μm, ultrasonic on 2 seconds and off 5 seconds). During the ultrasonication, the time was set to 1 minute, 2 minutes 30 seconds, 2 minutes 40 seconds, 2 minutes 50 seconds, 3 minutes 10 seconds, 3 minutes 20 seconds, 3 minutes 30 seconds, 4 minutes 30 seconds, 5 minutes, during which the cells were not overheated. After the disruption, adherent cells were washed with PBS 3 times to change serum-containing medium, the suspension cells were centrifuged to discard the supernatant, then resuspended with serum-containing medium and returned to the original wells, and then subjected to conventional cell culture for 24 hours. Then, whether the cells survive or not is observed, and the cell disruption efficiency is compared at different disruption times.
The results show that the samples of the two cells, whether the cells are adherent or suspended cells, are broken for 1 minute, and still have viable cells after 24 hours of culture. Samples of 2 min 30 sec, 2 min 40 sec, 2 min 50 sec, 3 min 10 sec, 3 min 20 sec, 3 min 30 sec, 4 min 30 sec, and 5 min were disrupted, and after 24 hours of incubation, no cell survival was seen in the wells of the 6-well plate.
Thus, the time for cell disruption is selected to be between 2 min 30 s and 3 min 30 s, preferably 2 min 40 s, 2 min 50 s, 3 min 10 s or 3 min 20 s, the cell disruption time being selected to ensure that the cells are sufficiently disrupted compared to 1 min of disruption; compared with the method for crushing for 5 minutes, the method avoids protein degradation caused by temperature rise of the protein solvent with long crushing time.
Example 2 search of spatial extent of cell disruption
MC3T3 osteoblasts, A375 melanoma cells at 5X10 per dish5The number of cells is respectively injected into culture dishes with the diameters of 60mm and 100mm, the temperature is 37 ℃, and the CO content is 5 percent2Culturing is carried out under the condition of serum-containing culture medium. After 24 hours, the cells were washed by adding 4mL or 8mL of PBS to each of 60mm and 100mm dishes, and the PBS was discarded. 30mL or 78.6mL PBS was added to a 60mm, 100mm petri dish. Adherent cells were disrupted by a cell disrupter (power 45W, frequency 21KHz, amplitude 35 μm, ultrasonic on 2 sec and off 5 sec) in ice bath for 3 min without stirring during ultrasonication. After the disruption, adherent cells were washed with PBS 3 times to change serum-containing medium, the suspension cells were centrifuged to discard the supernatant, then resuspended with serum-containing medium and returned to the original wells, and then cell culture was performed uniformly for 24 hours. Thereafter, the survival of the cells was observed, and the cell disruption efficiency of the 60mm and 100mm culture dishes were compared.
The results showed that adherent cells in a 60mm dish were disrupted by ultrasonication for 3 minutes, and no viable cells remained after 24 hours of culture. Adherent cells in a 100mm dish were disrupted for 3 minutes and very few cells remained on the edge of the dish after 24 hours of culture.
Thus, the critical spatial range for cell disruption is in the range of more than 30mm but less than 50mm from the center of the disruptor horn. The crushing operation is therefore carried out using culture vessels having a diameter of less than 100mm, preferably less than 60 mm.
Example 3 estimation of Total cellular protein concentration
The MC3T3 osteoblasts and A375 melanoma cells were plated at 5X 10/well5The cell number of (2) was injected into 6-well plates (diameter: 34.8mm), 37 ℃ and 5% CO2Culturing is carried out under the condition of serum-containing culture medium. After 24 hours, the cells in the 6-well plate were washed with 2ml PBS, and PBS was discarded. Adding 200 mu L of pancreatin into 3 wells of a 6-well plate to digest the cells, stopping digestion by using a serum-containing culture medium after 3 minutes, blowing the cells into single cells, washing the cells for three times by using PBS, resuspending the cells by 13mL of PBS, and putting the cells back into the respective wells; to the 6 hole plate in another 3 hole each adding 13 mLPBS. Adherent cells that were not subjected to trypsinization and suspended cells resuspended in PBS after trypsinization were disrupted for 3 minutes using a cell disrupter (power 45W, frequency 21KHz, amplitude 35 μm, ultrasonic on 2 seconds and off 5 seconds). No stirring was performed during ultrasonication. After disruption, 1ml of the liquid was centrifuged at 16000g for 10min at 4 ℃ and 20ul of the supernatant was collected, and the total protein concentration was measured by BCA kit (purchased from ThermoFisher Scientific).
The final estimation of total protein concentration of MC3T3 osteoblasts in 6-well plates per well was 20.79. + -. 3.57. mu.g/mL for suspension cells and 29.12. + -. 3.60. mu.g/mL for adherent cells (FIG. 1). The final estimate of total protein content per well cell was 270.21 + -46.43 μ g for suspension cells and 378.55 + -46.74 μ g for adherent cells (FIG. 2). The experimental results reveal that adherent cells have better disruption efficiency and the protein yield is higher compared with that of suspension cells.
The final estimation result of the total protein concentration of A375 melanoma cells in each well of 6-well plate is 25.79 +/-4.46 mu g/mL of suspension cells and 33.88 +/-4.31 mu g/mL of adherent cells (FIG. 3); the final estimates of total protein content per well cell were 335.21 + -57.99 μ g for suspension cells and 440.45 + -55.97 μ g for adherent cells (FIG. 4). The experimental result reveals that adherent cell ultrasound has better crushing efficiency, and the protein yield is higher than that of suspension cell ultrasound.
Compared with the method of crushing the adherent cells, the method of crushing the adherent cells greatly increases the total protein concentration, which means that the adherent cells are more fully exposed after being crushed by ultrasonic waves (fig. 1-4). Therefore, the total protein content of the adherent cells can be more accurately measured by carrying out ultrasonic disruption after the adherent treatment of the cells and fully extracting the total protein of the adherent cells.
Example 4
Total protein content of human cardiac microvascular endothelial cells (purchased from Sciencell, USA) after cell attachment and suspension sonication was measured as in example 3 with a cell plating density of 50% at sonication. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 5
Total protein content of human cortical neuronal cells (purchased from Wuhan Protocet Life technologies, Inc.) after cell attachment and suspension sonication was examined as in example 3, except that the cell plating density at sonication was 90%. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 6
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set at a power of 30W, a frequency of 30KHz, an amplitude of 25 μm, 5 seconds for ultrasound on/off for 8 seconds, and a disruption time of 3 minutes and 30 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 7
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set at a power of 60W, a frequency of 15KHz, an amplitude of 40 μm, an ultrasonic on/off time of 1 second for 4 seconds, and a disruption time of 2 minutes and 30 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 8
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set to a power of 55W, a frequency of 18KHz, an amplitude of 37 μm, a sonication on/off time of 3 seconds for 6 seconds, and a disruption time of 2 minutes and 40 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 9
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set at a power of 50W, a frequency of 20KHz, an amplitude of 34 μm, 4 seconds for ultrasonic on/off 7 seconds, and a disruption time of 2 minutes 50 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 10
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set at a power of 40W, a frequency of 22KHz, an amplitude of 32 μm, 2 seconds on and off with 5 seconds on sonication, and a disruption time of 2 minutes and 30 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 11
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set at 35W power, 24KHz frequency, 30 μm amplitude, 2 seconds on and off for 5 seconds with sonication time of 2 minutes and 30 seconds. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 12
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set to 45W power, 15KHz frequency, 40 μm amplitude, 5 seconds on/off of the sonication for 8 seconds, and 2 minutes/30 seconds off. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
Example 13
The total protein content of MC3T3 after osteoblast adherence and suspension sonication was determined as in example 3, except that the sonication parameters were set to 45W power, 30KHz frequency, 25 μm amplitude, 1 second ultrasonic on/off for 4 seconds, and 2 minutes 30 seconds disruption time. The results show that the total protein concentration of the cell adherent disruption method is greatly increased compared with the suspension cell disrupted by ultrasonic wave, which indicates that the method can fully extract the total protein of the adherent cell and can more accurately measure the total protein content of the cell.
It should also be noted that, in the case of the embodiments of the present invention, features of the embodiments and examples may be combined with each other to obtain a new embodiment without conflict.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and the scope of the present invention is subject to the scope of the claims.
Claims (10)
1. A method for extracting total protein from adherent cells, comprising performing ultrasonic lysis for cells in their adherent state, wherein the conditions of the ultrasonic lysis are as follows: the time is from 2 minutes 30 seconds to 3 minutes 30 seconds, preferably 2 minutes 40 seconds, 2 minutes 50 seconds, 3 minutes 10 seconds or 3 minutes 20 seconds,
the power is 30W-60W, for example 35W, 40W, 45W, 50W or 55W, preferably 45W,
the frequency is 15KHz-30KHz, such as 18KHz, 19KHz, 20KHz, 21KHz, 22KHz, 23KHz, 24KHz, 25KHz or 28KHz, preferably 21KHz,
an amplitude of 25-40 μm, for example 27 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 37 μm or 39 μm, preferably 35 μm,
ultrasound is switched on for 1-5 seconds and off for 4-8 seconds, and ultrasound is cycled, for example, ultrasound is switched on for 2 seconds and off for 5 seconds, ultrasound is switched on for 3 seconds and off for 6 seconds, or ultrasound is switched on for 4 seconds and off for 7 seconds, and ultrasound is preferably switched on for 2 seconds and off for 5 seconds.
2. The method of claim 1, comprising adherent culture and sonication of adherent cells in a culture vessel having a diameter of less than 100mm, preferably less than 60 mm.
3. The method of any one of claims 1-2, wherein the cell buffer used in the sonication lysis is a protein-free cell buffer.
4. The method of claim 3, wherein the protein-free cell buffer is PBS.
5. The method of claim 3 or 4, wherein the level of the cell buffer used in the culture dish is greater than 1cm during the ultrasonication.
6. The method of any one of the above claims, wherein the sonication is performed under ice bath conditions.
7. The method of any of the preceding claims, wherein the cell is a cell capable of adherent growth, e.g. a fibroblast-type cell, such as a fibroblast, cardiomyocyte, smooth muscle cell, osteoblast or vascular endothelial cell (e.g. human cardiac microvascular endothelial cell), an epithelial cell-type cell, such as a skin epithelial cell, an alimentary epithelial cell, a mammary epithelial cell, an alveolar epithelial tumor cell (e.g. melanoma cell, ovarian cancer cell), a migratory cell-type cell, such as a highly differentiated nasopharyngeal cancer cell, a polymorphous cell-type cell, such as a neural cell (e.g. neuronal cell).
8. The method of any of the above claims, wherein the cells are sonicated at a cell density of 50% to 100% of the confluent culture dish, e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.
9. The method of any of the above claims, wherein the method does not include a stirring step upon sonication.
10. A method for accurately determining total protein of adherent cells, comprising extracting total protein of adherent cells according to the method for extracting total protein of adherent cells of any one of the preceding claims, and determining the concentration of total protein using BCA assay.
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| CN117442714A (en) * | 2023-12-26 | 2024-01-26 | 成都柏奥特克生物科技股份有限公司 | Novel varicella attenuated live vaccine and preparation method thereof |
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