阅读说明：本技术 一种蛋白质提取试剂及其制备方法以及蛋白质提取方法 (Protein extraction reagent, preparation method thereof and protein extraction method ) 是由 张煜 于 2021-06-19 设计创作，主要内容包括：本发明公开了一种蛋白质提取试剂及其制备方法以及蛋白质提取方法,蛋白质提取试剂包含质量体积比为40g/L的尿素、质量体积比为1g/L的十二烷基硫酸钠、体积百分含量为0.1％的聚乙二醇辛基苯基醚、质量体积比为8g/L的氯化钠、质量体积比为0.2g/L的氯化钾、质量体积比为1.42g/L的磷酸氢二钠、质量体积比为0.27g/L的磷酸二氢钾、质量体积比为8g/L的乙二胺四乙酸、体积百分含量为1％的二甲基亚砜；制备方法包含将乙二胺四乙酸粉末溶解于800mL去离子水后加40g尿素溶解,加十二烷基硫酸钠、聚乙二醇辛基苯基醚、氯化钠、氯化钾、磷酸氢二钠、磷酸二氢钾、二甲基亚砜溶解定容至1升制得蛋白质提取试剂。(The invention discloses a protein extraction reagent, a preparation method thereof and a protein extraction method, wherein the protein extraction reagent comprises 40g/L of urea, 1g/L of sodium dodecyl sulfate, 0.1% of polyethylene glycol octyl phenyl ether, 8g/L of sodium chloride, 0.2g/L of potassium chloride, 1.42g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate, 8g/L of ethylene diamine tetraacetic acid and 1% of dimethyl sulfoxide; the preparation method comprises the steps of dissolving ethylene diamine tetraacetic acid powder in 800mL of deionized water, adding 40g of urea for dissolving, adding sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate and dimethyl sulfoxide for dissolving and fixing the volume to 1 liter to obtain the protein extraction reagent.)

1. A protein extraction reagent is characterized by comprising urea, sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, ethylene diamine tetraacetic acid and dimethyl sulfoxide.

2. The protein extraction reagent according to claim 1, wherein the urea has a mass-to-volume ratio of 40 g/L;

the mass volume ratio of the sodium dodecyl sulfate is 1 g/L;

the volume percentage content of the polyethylene glycol octyl phenyl ether is 0.1 percent;

the mass volume ratio of the sodium chloride is 8 g/L;

the mass volume ratio of the potassium chloride is 0.2 g/L;

the mass-to-volume ratio of the disodium hydrogen phosphate is 1.42 g/L;

the mass volume ratio of the potassium dihydrogen phosphate is 0.27 g/L;

the mass volume ratio of the ethylene diamine tetraacetic acid is 8 g/L;

the volume percentage of the dimethyl sulfoxide is 1 percent.

3. The protein extraction reagent of claim 2, further comprising concentrated hydrochloric acid, wherein the pH of said protein extraction reagent is adjusted to 7.0-7.5 using said concentrated hydrochloric acid.

4. A method for preparing a protein extraction reagent, comprising the steps of:

step A1: dissolving ethylenediamine tetraacetic acid powder in 800mL of deionized water;

step A2: adding 40g of urea for dissolution;

step A3: and sequentially adding sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate and dimethyl sulfoxide, dissolving, and metering the volume to 1000mL to obtain the protein extraction reagent.

5. The method of claim 4, wherein the pH of the protein extraction reagent obtained in step A3 is adjusted to 7.0-7.5 by adding concentrated hydrochloric acid, and then deionized water is added to 1L, followed by filtration sterilization using 0.2 μm filter.

6. A protein extraction method is characterized by comprising the following steps:

step B1: obtaining a tissue, placing the tissue in phosphate buffer saline solution for washing three times, and removing redundant blood or culture solution;

step B2: adding 100mg of tissue or cells into a centrifuge tube according to the proportion of 1ml of protein extraction reagent, adding grinding ceramic beads, and grinding in a tissue homogenizer;

step B3: after completion of the milling, the suspension was left on ice for 5min, followed by centrifugation to obtain a total protein solubilized supernatant.

7. The protein extraction method as claimed in claim 6, wherein 3-7 ceramic beads are added in step B2 and ground in a tissue homogenizer at 4 deg.C for 2 min.

8. The method for extracting protein according to claim 7, wherein the centrifugation in step B3 is performed at 12000rpm for 5 min.

9. Use of a protein extraction reagent according to any one of claims 1 to 3 in protein polyacrylamide gel electrophoresis, protein Western blot qualitative and/or quantitative, immunoprecipitation and co-immunoprecipitation.

Technical Field

The invention relates to the field of protein extraction reagents, in particular to a protein extraction reagent, a preparation method thereof and a protein extraction method.

Background

Proteomics research can reveal life rules by constructing time and space difference maps of protein expression in cells, the SDS polyacrylamide gel electrophoresis separation technology of protein is the key technology of proteomics research, and the key technology of proteomics difference analysis is whether total protein is completely extracted, in the extraction of total protein, cell lysate is a reagent widely used in protein analysis and detection, and is divided into strong, medium and weak due to the intensity of lysed cells or tissues, and is respectively used in different applications, however, the three cell tissue lysates can not completely extract total protein in cells or tissues, and due to the limitation of conditions, cell precipitation or a large amount of lipoprotein and the like in the lysate can often appear;

in recent years, protein extraction, such as cell nucleus protein extraction, cell membrane protein extraction, cytoplasm protein extraction, mitochondrial protein extraction and the like, is more specialized and is respectively used in different scientific research fields, but the technologies still have the defect that total protein cannot be completely extracted;

the RIPA total protein extraction method is widely applied to proteomics research by the characteristics of low price, simple operation, good repeatability and the like, and widely used reagents comprise sodium dodecyl sulfate, dimethyl sulfoxide, triton and the like, which can play roles of cracking cells, promoting protein dissolution, preventing protein degradation in cell lysate and the like, so that the extraction of total protein in cells or tissues is greatly improved, but the pretreatment steps of protein extraction are more complicated, the operation time is greatly prolonged, and the probability of tissue or cell pollution is increased.

Disclosure of Invention

The invention provides a protein extraction reagent and a preparation method of the protein extraction reagent, and also provides a protein extraction method, which can be used for shortening the time for extracting the whole protein in the tissue or the cell without pretreating the cell or the tissue before extraction, greatly reducing the possibility of polluting a protein sample, greatly improving the extraction efficiency of the total protein in the cell and the tissue and having lower cost; the extraction of protein can be completed in a very short time according to the use method of the protein extraction reagent, the whole protein in cells or tissues can be obtained in a short time, the added urea and the added polyethylene glycol octyl phenyl ether can effectively crack the cells and the tissues to promote the dissolution of the protein, and the added ethylene diamine tetraacetic acid and the added dimethyl sulfoxide are beneficial to inhibiting the degradation of the protein, can extract the protein with lower expression level in the tissues or the cells without reducing the activity of the protein, and are used for solving the defects caused by the prior art.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, a protein extraction reagent comprises urea, sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, ethylene diamine tetraacetic acid, and dimethyl sulfoxide.

The protein extraction reagent is characterized in that the mass-to-volume ratio of the urea is 40 g/L;

the mass volume ratio of the sodium dodecyl sulfate is 1 g/L;

the volume percentage content of the polyethylene glycol octyl phenyl ether is 0.1 percent;

the mass volume ratio of the sodium chloride is 8 g/L;

the mass volume ratio of the potassium chloride is 0.2 g/L;

the mass-to-volume ratio of the disodium hydrogen phosphate is 1.42 g/L;

the mass volume ratio of the potassium dihydrogen phosphate is 0.27 g/L;

the mass volume ratio of the ethylene diamine tetraacetic acid is 8 g/L;

the volume percentage of the dimethyl sulfoxide is 1 percent.

The protein extraction reagent further comprises concentrated hydrochloric acid, and the pH value of the protein extraction reagent is adjusted to 7.0-7.5 by using the concentrated hydrochloric acid.

In a second aspect, a method for preparing a protein extraction reagent, comprising the steps of:

step A1: dissolving ethylenediamine tetraacetic acid powder in 800mL of deionized water;

step A2: adding 40g of urea for dissolution;

step A3: and sequentially adding sodium dodecyl sulfate, polyethylene glycol octyl phenyl ether, sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate and dimethyl sulfoxide, dissolving, and metering the volume to 1000mL to obtain the protein extraction reagent.

In the above method for preparing a protein extraction reagent, concentrated hydrochloric acid is added to the protein extraction reagent obtained in step a3 to adjust the pH to 7.0-7.5, and then deionized water is added to a volume of 1L, followed by filtration sterilization using a 0.2 μm filter membrane.

In a third aspect, a method for protein extraction, comprising the steps of:

step B1: obtaining a tissue, placing the tissue in phosphate buffer saline solution for washing three times, and removing redundant blood or culture solution;

step B2: adding 100mg of tissue or cells into a centrifuge tube according to the proportion of 1ml of protein extraction reagent, adding grinding ceramic beads, and grinding in a tissue homogenizer;

step B3: after completion of the milling, the suspension was left on ice for 5min, followed by centrifugation to obtain a total protein solubilized supernatant.

In the above protein extraction method, 3-7 ceramic beads are added in step B2 and ground in a tissue homogenizer at 4 deg.C for 2 min.

In the above protein extraction method, the centrifugation in step B3 is performed at 12000rpm for 5 min.

In a fourth aspect, the protein extraction reagent provided in the first aspect is applied to protein polyacrylamide gel electrophoresis, protein Western blot qualitative and/or quantitative analysis, immunoprecipitation and co-immunoprecipitation.

The technical scheme provided by the protein extraction reagent, the preparation method thereof and the protein extraction method has the following technical effects:

the protein extraction reagent provided by the invention does not need to pretreat cells or tissues before extraction, can shorten the time for extracting the whole protein in the tissues or cells, greatly reduces the possibility of pollution of protein samples, can greatly improve the extraction efficiency of the total protein in the cells and the tissues, and has lower cost;

the extraction of protein can be completed in a very short time according to the use method of the protein extraction reagent, the whole protein in cells or tissues can be extracted in a short time, the added urea and the added polyethylene glycol octyl phenyl ether can effectively crack the cells and the tissues to promote the dissolution of the protein, and the added ethylene diamine tetraacetic acid and the added dimethyl sulfoxide are beneficial to inhibiting the degradation of the protein, so that the protein with low expression level in the tissues or the cells can be extracted without reducing the activity of the protein.

Drawings

FIG. 1 is a graph showing the results of WB detection and GAPDH detection as an index for each sample;

FIG. 2 is a graph showing the WB detection results of each sample, and the detection index is β -actin.

Detailed Description

In order to make the technical means, the inventive features, the objectives and the effects of the invention easily understood and appreciated, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the specific drawings, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments.

All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention.

In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.

The invention provides a protein extraction reagent, a preparation method thereof and a protein extraction method, aiming at not needing to pre-treat cells or tissues before extraction, shortening the time for extracting tissues or total proteins in the cells, greatly reducing the possibility of pollution of protein samples, greatly improving the extraction efficiency of the total proteins in the cells and the tissues and having lower cost; the extraction of protein can be completed in a very short time according to the use method of the protein extraction reagent, the whole protein in cells or tissues can be extracted in a short time, the added urea and the added polyethylene glycol octyl phenyl ether can effectively crack the cells and the tissues to promote the dissolution of the protein, and the added ethylene diamine tetraacetic acid and the added dimethyl sulfoxide are beneficial to inhibiting the degradation of the protein, so that the protein with low expression level in the tissues or the cells can be extracted without reducing the activity of the protein.

Preparing a protein extraction reagent, dissolving ethylenediaminetetraacetic acid powder in 800mL of deionized water to obtain a solution with the mass-volume ratio of 8g/L, then adding 40g of urea for dissolution, then sequentially adding 1g/L of lauryl sodium sulfate, 0.1% of polyethylene glycol octyl phenyl ether, 8g/L of sodium chloride, 0.2g/L of potassium chloride, 1.42g/L of disodium hydrogen phosphate, 0.27g/L of potassium dihydrogen phosphate and 1% of dimethyl sulfoxide, dissolving and fixing the volume to 1000mL to obtain the protein extraction reagent;

then adding concentrated hydrochloric acid into the protein extraction reagent to adjust the pH value to 7.0-7.5, then adding deionized water to a constant volume of 1L, and performing filtration sterilization by using a 0.2 mu m filter membrane;

obtaining tissues, namely adopting 330mg of rat liver, dividing the tissues into a group A, a group B, a group C and a group D, adopting 50mg of rat liver for each group of tissues, putting the tissues in the group A, the group B, the group C and the group D into phosphate buffer saline solution, washing for three times, removing redundant blood or culture solution, operating the group A by using a bionate, operating the group B by using solarbio, operating the group C by using the bionate, adding phenylmethylsulfonyl fluoride, operating the group D by using solarbio and adding phenylmethylsulfonyl fluoride;

respectively adding 0.5ml of protein extraction reagent into the A group, B group, C group and D group, adding 3-7 grinding ceramic beads, and grinding at 4 deg.C for 2 min;

after completion of the milling, the resultant was left to stand on ice for 5min and then centrifuged at 12000rpm for 5min to obtain supernatant of total protein solubilization in groups A, B, C and D.

The obtained total protein of group A, group B, group C and group D dissolved supernatant was subjected to WB detection with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and β -actin (actin) as detection indexes, and the results are shown in FIGS. 1-2.

In conclusion, the protein extraction reagent, the preparation method thereof and the protein extraction method provided by the invention can be used for shortening the time for extracting the tissue or the whole protein in the cell without pretreating the cell or the tissue before extraction, greatly reducing the possibility of pollution of a protein sample, greatly improving the extraction efficiency of the total protein in the cell and the tissue, and being low in cost; the extraction of protein can be completed in a very short time according to the use method of the protein extraction reagent, the whole protein in cells or tissues can be extracted in a short time, the added urea and the added polyethylene glycol octyl phenyl ether can effectively crack the cells and the tissues to promote the dissolution of the protein, and the added ethylene diamine tetraacetic acid and the added dimethyl sulfoxide are beneficial to inhibiting the degradation of the protein, so that the protein with low expression level in the tissues or the cells can be extracted without reducing the activity of the protein.

Specific embodiments of the invention have been described above. It is to be understood that the invention is not limited to the particular embodiments described above, in that devices and structures not described in detail are understood to be implemented in a manner common in the art; various changes or modifications may be made by one skilled in the art within the scope of the claims without departing from the spirit of the invention, and without affecting the spirit of the invention.