CN108727464A - A kind of new Protein Extraction Reagent and method - Google Patents

Abstract

The invention provides a protein extraction reagent, which includes the following components per 10ml of aqueous solution: Tris 14.5-15.5mg, glycine 74-75mg, SDS 202-208mg, bromophenol blue 10-12mg, Tris-HCl 0.5-0.6ml, Glycerin 0.8~1.2ml, β-mercaptoethanol 0.4-0.6ml. The invention not only solves the detection problem of trace cells, but also improves the detection sensitivity of Western blot.

Description

A new protein extraction reagent and method

technical field

The invention belongs to the field of biotechnology and relates to protein extraction.

Background technique

Western blot (Western blot) is the most important experimental method for protein detection in the scientific research industry. As long as protein detection is involved, Western blot must be the first choice. Western blot is an experimental method commonly used in molecular biology, biochemistry and immunogenetics. The basic principle is to stain the gel electrophoresis-treated cells or biological tissue protein samples through specific antibodies. Information about the expression of specific proteins in the analyzed cells or tissues can be obtained by analyzing the location and depth of staining. It is the most commonly used and most important protein detection technique. However, before performing Western blot, the total protein of cells or biological tissues must be extracted. Western blot is an experimental technique that uses the extracted total protein as the detection object.

Generally speaking, the total protein of biological tissue is easier to extract, because the amount of tissue is enough, so the total protein extracted is large enough and the total protein concentration is high enough. But the total protein extraction of cultured cells is not so easy. Because Westernblot has requirements for the total protein amount and concentration extracted. The amount of protein in cultured cells is far less than that in tissues. Even if the total protein can be extracted by adding traditional lysate, the protein concentration is often not enough to be detected by Western blot. At this time, if you want to increase the protein concentration, you can reduce the amount of lysate, but after reducing the lysate, the total protein amount extracted will decrease, and the Western blot experiment cannot be performed if the concentration reaches the insufficient protein amount. Therefore, in order to ensure the protein amount and concentration of cultured cells. Just increase the number of cultured cells. In the traditional experimental method, in the best case, at least 2 wells of a 6-well plate are required to have enough protein for Western blot.

In most practical operations, the amount of protein in 3 to 4 wells of a 6-well plate is generally used. This increase in the amount of cells will most directly increase our experimental material costs and labor costs. Secondly, this situation will consume more resources and generate more garbage and harmful products, which is extremely detrimental to environmental protection. Furthermore, due to the large amount of cells required, it is inevitable to increase operational errors when processing the cells, which will affect the experimental results. Finally, and most importantly, for many kinds of cells, especially primary cells, rare cells, newly cultivated cells, precious cells, non-proliferating cells, etc., we cannot obtain the amount of protein required by traditional protein extraction at all during experiments, and cannot Perform Western blot. So we urgently need a new and efficient method for extracting total protein to solve these problems.

Contents of the invention

The purpose of the present invention is to provide a new protein extraction reagent, which not only solves the detection problem of trace cells, but also improves the detection sensitivity of Western blot.

The object of the present invention is achieved by the following measures:

A protein extraction reagent, including the following components per 10ml of aqueous solution: Tris (trishydroxymethylaminomethane) 14.5-15.5mg, glycine 74-75mg, SDS 202-208mg, bromophenol blue 10-12mg, Tris-HCl 0.5 ～0.6ml, glycerin 0.8～1.2ml, β-mercaptoethanol 0.4-0.6ml.

Preferably, the optimal ratio of each component in the above protein extraction reagent is: each 10ml aqueous solution contains the following components: Tris 15mg, glycine 75mg, SDS 205mg, bromophenol blue 10mg, Tris-HCl of pH 6.8 0.5ml , glycerol 1ml, β-mercaptoethanol 0.5ml.

The dosage of the above protein extraction reagent is 400 μL-600 μL per 1×10 ⁶ cells; or 200 μL-300 μL per well of a 24-well plate.

Another object of the present invention is to provide a method for extracting protein using the above reagents.

A protein extraction method, comprising the following steps:

(1) Collect the cells, add 400 μL to 600 μL of the protein extraction reagent per 1×10 ⁶ cells after washing;

(2) Stand still for 30 seconds to 2 minutes;

(3) Boil for 10-20 minutes to denature the protein, then cool.

Specifically, the method for using the above-mentioned protein extraction reagent includes the following steps:

(1) Prepare protein extraction reagent: add Tris, glycine, SDS, bromophenol blue, Tris-HCl, glycerol, β-mercaptoethanol, water in proportion;

(2) For adherent cells: remove the culture medium, wash with buffer solution, add 200 μL to 300 μL of the above reagent per well of a 24-well plate; gently blow and mix to make the extract fully contact with the cells; for suspension cells: centrifuge Collect the cells, break up the cells, add 200 μL to 300 μL to each well of the 24-well plate, and gently blow and mix to make the extract fully contact with the cells;

(3) Heating at 100°C or in a boiling water bath for 15 minutes to fully denature the protein; store the denatured protein at -20°C until western loading. The loading volume of each well of SDS-PAGE was set at 20 microliters. Samples can be stored for one year.

According to the above steps, 10 westerns can be performed per well of a 24-well plate. In conversion, the new method only needs one-eightyth of the protein amount of the traditional method.

Beneficial effect

1. The biggest problem with traditional protein extraction is that a large number of cells need to be cultured, but not all cells can be cultured in large quantities, and the traditional method cannot extract effective total protein for a small amount of cells. However, the present invention only needs one eightyth of the protein amount of the traditional method, which not only solves the detection problem of trace cells, but also improves the detection sensitivity of Western blot.

2. The invention is not only helpful to cells that cannot be cultured in large quantities, but also improves the sensitivity of protein detection; it is also helpful to the detection of protein in normally cultured cells. First of all, due to the small amount of cells, a detection group only needs cells in one well of a 24-well plate, so it can reduce the cultivation of cells, saving time and money costs; The new protein sample solution is enough to do 10 times of Western blot, which ensures the amount of repeated detection and improves the detection accuracy of Western blot.

3. The steps of the present invention are simple and easy to operate, and it only takes a few minutes to complete the extraction. The invention simplifies the experimental steps and reduces the experimental operation error rate. For the same sample processing, the present invention only needs one or two minutes in the protein cleavage link, while the old method needs to operate on ice for more than 30 minutes, and the time is greatly reduced. Due to the reduction of time, there is no need to operate on ice all the time, which simplifies the experimental preparation.

4. In traditional protein extraction, some operators will detect the concentration of the protein after protein extraction, and then adjust the amount of lysate according to the concentration, so that the total protein loaded in different groups of the same batch remains at the same concentration, so that the Western blot will run out The internal reference β-actin between different samples is theoretically consistent, and the difference of the target protein is relatively clear at a glance. Its advantage is that it is convenient to judge the results by visually viewing the graph, but the quantitative analysis results are not necessarily related to this operation. And in actual operation, it is still impossible to achieve the same internal reference, and it often comes out uneven. For this difference, the inventor provides two reference solutions. The first solution is to directly measure the protein concentration with an OD-1000 or OD-2000 spectrophotometer after the protein is extracted by the new method, so as to determine the loading amount and unify the internal reference. But the inventor recommends option two more. Scheme 2 directly subtracts the step of protein concentration detection, reduces operation steps and time, and uses another method to maintain the unity of the internal reference. The specific steps are to count the cells when the cells are cultured and plated, so that the number of cells planted in each well is the same, then the total protein amount of the cells in each well is determined to be the same when the cells are planted, and the internal reference that comes out is approximately the same. If there is a big difference in the internal reference of the protein released, you can adjust the sample volume to make the internal reference neat, making the results easier to observe.

5. The protein of the present invention is completely extracted, from 15KD to 22KD as an example, can be completely extracted and detected, and can completely replace traditional reagents and methods.

6. The present invention does not have high requirements on the cell state. Whether it is drug addition or physical and mechanical treatment, protein can be extracted efficiently and completely. In the actual experiment, after various treatments, the state of the cells is good or bad, and many of the proliferative ability begins to decline, and even the cells begin to senescence and apoptosis intensifies, but the present invention can efficiently and completely extract the cells no matter what state.

7. The present invention is applicable to various cells. Both human-derived cells and mouse-derived cells can be extracted. Even primary cells, which are difficult to cultivate, can still extract proteins intact.

8. The efficiency of the present invention is higher. The traditional method requires a large amount of original cells; while the present invention requires a low amount of cells. The traditional method is to strip the protein from the cells, and the extraction is incomplete; the present invention does not need to separate the protein and other components of the cell, the obtained protein is more complete, and the experimental results are more accurate and effective. The extraction amount of the traditional method is not high; the extraction amount of the present invention is sufficient.

Description of drawings

Fig. 1 The present invention is used for stimulating human umbilical cord mesenchymal stem cells by IFN-γ

Figure 2 The present invention uses LIPUS and 2-APB to act on mouse bone marrow mesenchymal stem cells

Figure 3 β-actin compares the difference in protein extraction effect between the old and new methods

Detailed ways

The present invention is specifically described below through the examples, it is necessary to point out that the following examples are only used to further illustrate the present invention, and can not be interpreted as limiting the protection scope of the present invention, those skilled in the art can according to the content of the present invention above Some non-essential improvements and adjustments are made to the present invention.

Example 1

A protein extraction reagent, weighing Tris 14.5~15.5mg, glycine 74~75mg, SDS 205mg, bromophenol blue 10~12mg, Tris-HCI (pH6.8) 0.5~0.6ml, glycerol 0.8~1.2ml, β- Mercaptoethanol 0.5ml, add deionized water to make up to 10ml. It can be stored for one year at -20°C.

1. The protein extraction reagent is used for IFN-γ stimulation of human umbilical cord mesenchymal stem cells

(1) Isolation of primary human umbilical cord mesenchymal stem cells. Cultured to the third generation of primary human umbilical cord mesenchymal stem cells.

( ² ) Add 1x105 human umbilical cord mesenchymal stem cells to each well of a 24-well plate, and then add different concentrations of IFN-γ (a drug that can stimulate target cells to secrete related protein factors) after adhering to the wall. The concentrations of IFN-γ are respectively 0ng/ml, 20ng/ml, 5ng/ml0, 100ng/ml. Cellular proteins were ready to be harvested after 48 hours of culture.

(3) Remove the medium, wash with buffer three times, add 200 μL of protein extraction reagent to each well of a 24-well plate, gently blow and mix, let stand for 1 minute, and put the liquid into an EP tube. Boil for 15 minutes to denature the egg whites. After cooling, store at -20°C.

(4) After the protein was extracted, Western blot was used to detect the expression level of the target protein molecule in the human umbilical cord mesenchymal stem cells. Proteins β-actin, LC3, P62, Beclin were detected. Take 20uL protein from each group, transfer the protein to PVDF membrane by electroporation, wash 5 times with TBST, 3min each time; ), incubated overnight at 4°C, washed 5 times with TBST, 3 min each time, then added secondary antibody, incubated at room temperature for 4 h, washed 5 times with TBST, 3 min each time, and developed color by chemiluminescence.

The experimental results are shown in Figure 1. Among them, the molecular weight of β-actin protein is 45KD, the molecular weight of LC3 protein is 15KD, the molecular weight of P62 protein is 62KD, and the molecular weight of Beclin protein is 60KD. The molecular weight of protein is generally between 10 and 250KD, LC3 molecular weight of 15KD can represent the extraction ability of small molecular weight proteins, and other proteins can represent the extraction ability of medium molecular weight proteins. (anti-LC3 antibody US CST company, anti-Beclin1 antibody US abcam company, anti-P62 antibody US sigma company, β-actin US sigma company)

2. The protein extraction reagent is used for LIPUS to act on mouse bone marrow mesenchymal stem cells

(1) Isolation of primary mouse bone marrow mesenchymal stem cells. Cultivated to the third generation.

( ² ) 1x105 mouse bone marrow mesenchymal stem cells were added to each well of a 24-well plate, and the cells were treated with low-intensity focused ultrasound (LIPUS) after attachment, and cell proteins were ready to be collected after 48 hours of culture.

(3) Remove the culture medium, wash with the buffer solution 3 times, add 200 μL extract solution to each well of the 24-well plate, gently blow and mix, let stand for 1 minute, and put the liquid into the EP tube. Boil for 15 minutes to denature the egg whites. After cooling, store at -20°C.

(4) After the protein was extracted, Western blot was used to detect the expression level of the target protein molecule in the mouse bone marrow mesenchymal stem cells. The proteins GAPDH, TRPM7 were detected. Take 20uL protein from each group, transfer the protein to PVDF membrane by electroporation, wash 5 times with TBST, 3min each time; block with 5% skimmed milk powder at room temperature for 1h, add GAPDH, TRPM7 antibody (1:1000 dilution), incubate overnight at 4°C, Wash 5 times with TBST, 3 min each time, then add secondary antibody, incubate at room temperature for 4 h, wash 5 times with TBST, 3 min each time, develop color by chemiluminescence.

The experimental results are shown in Figure 2. Figure 2 shows the effect of LIPUS on mouse bone marrow mesenchymal stem cells. The detected molecular weight of Trpm7 protein is 220KD, and the molecular weight of GAPDH protein is 36KD. The molecular weight of protein is generally between 10 and 250KD, and the molecular weight of TRPM7 is 220KD, which can represent the extraction ability of large molecular weight proteins. (GAPDH US Santa Cruz, TRPM7 UK Abcam)

3. The protein extraction reagent extracts protein difference by β-actin comparing old and new methods

(1) Isolation of primary human umbilical cord mesenchymal stem cells. Cultured to the third generation of primary human umbilical cord mesenchymal stem cells.

( ² ) ^8x105 human umbilical cord mesenchymal stem cells were added to a T25 culture flask; 1x105 human umbilical cord mesenchymal stem cells were added to each well of a 24-well plate. Cellular proteins were ready to be harvested after 48 hours of culture.

(3) The method of the present invention: remove the culture medium, wash with buffer 3 times, add 200 μL of the protein extraction reagent described in Example 1 to each well of a 24-well plate, gently blow and beat to mix, stand still for 1 minute, and put the liquid into an EP tube.

Traditional method: extract the total protein in a T25 culture flask, the operation is as follows: because the extraction time is too long to prevent protein degradation, the whole process is operated on ice. Thaw RIPA lysate and mix well. Take an appropriate amount of lysate, and add PMSF (protease inhibitor) within a few minutes before use, so that the final concentration of PMSF is 1 mM. Remove culture medium and wash three times with buffer. Add lysate according to the ratio of adding 40 microliters of lysate to each well of a 6-well plate, that is, add 100 microliters of lysate to each bottle of T25. Pipet several times with a gun to make the lysate fully contact with the cells. Mix and shake, place on ice to lyse, shake and mix repeatedly every 10 minutes, a total of 3 times. After fully lysed, centrifuge at 10,000 g for 5 minutes, and take 80 microliters of supernatant. Dissolve SDS-PAGE loading buffer (5X). Add 20 μl SDS-PAGE loading buffer (5X) to 80 μl lysate. Wherein, all required reagents RIPA lysate, PMSF, and SDS-PAGE loading buffer (5X) are commercially available.

(4) Boil for 15 minutes to denature the protein. After cooling, store at -20°C.

(5) After protein extraction, Western blot was used to detect β-actin in human umbilical cord mesenchymal stem cells, and the loading volume was 20 microliters.

The results of the WB experiment are shown in Figure 3. (1) According to the traditional method, add 40 microliters of lysate to each well of a 6-well plate in the best case to obtain 50 microliters of samples, and the sample volume is 20 microliters, that is, 2 wells of a 6-well plate can be used for up to 5 westerns. However, each well of the 24-well plate of the present invention can perform 10 westerns. In conversion, the present invention only needs one-eightyth of the protein amount of the traditional method. (2) Figure 3A is a comparison between the traditional method of extracting a T25 protein and the new method of extracting β-actin from a well of a 24-well plate. The comparison of the amount of cells collected by the traditional method and the method of the present invention is 10:1. Figure 3B is the grayscale analysis value of β-actin contained in the WB strip. The data shows that the method of the present invention has only one-tenth of the cell mass of the traditional method, and the β-actin expression of the protein extracted by the present invention is more than three times that of the traditional method.

Claims (5)

1. A protein extraction reagent, including the following components in every 10ml aqueous solution: Tris14.5~15.5mg, glycine 74~75mg, SDS 202-208mg, bromophenol blue 10~12mg, Tris-HCl 0.5~0.6ml, glycerol 0.8~1.2ml, 0.4-0.6ml of β-mercaptoethanol. 2. protein extraction reagent as claimed in claim 1, every 10ml aqueous solution contains Tris-HCl 0.5ml, glycerol 1ml, β-mercaptoethanol 0.5ml of Tris 15mg, glycine 75mg, SDS205mg, bromophenol blue 10mg, pH6.8. 3. The protein extraction reagent according to claim 1 or 2, the dosage is to add 400 μL to 600 μL of the reagent per 1×10 ⁶ cells; or add 200 μL to 300 μL of the reagent to each well of a 24-well plate. 4. adopt the method for protein extraction described in claim 1,2 or 3 protein extraction reagents, comprise the following steps: (1) Collect the cells, add 400μL~600μL of the protein extraction reagent per ^1x106 cells after washing; (2) Stand still for 30 seconds to 2 minutes; (3) Boil for 10-20 minutes to denature the protein and cool down. 5. the method for extracting protein as claimed in claim 4, comprises the following steps: (1) Prepare protein extraction reagents: add Tris, glycine, SDS, bromophenol blue, Tris-HCl, glycerin, β-mercaptoethanol, and water in proportion; (2) For adherent cells: remove the culture medium, wash with buffer solution, add 200μL~300μL of the above reagent to each well of a 24-well plate; gently blow and mix to make the extract fully contact with the cells; For suspension cells: collect the cells by centrifugation, break up the cells, add 200μL~300μL to each well of the 24-well plate, and gently blow and mix to make the extract fully contact with the cells; (3) Heat at 100°C or in a boiling water bath for 15 minutes to fully denature the protein; store the denatured protein at -20°C for later use.