Extracting solution for extracting protein from adipose tissue and extracting method

Abstract

The invention provides an extracting solution for extracting protein from adipose tissue and an extracting method, so as to make up the defects in the prior art. The extracting solution is a PBS (Phosphate Buffer Solution) added with cane sugar, EDTA (Ethylene Diamine Tetraacetic Acid), SDS (Sodium Dodecyl Sulfate), sodium deoxycholate and Triton X-100; wherein the concentrations of the sucrose, the EDTA, the SDS, the sodium deoxycholate and the Triton X-100 are as follows: 50 mM of the PBS, 0.25 M of the sucrose, 0.5 mM of the EDTA, 0.5 mM of the SDS, 0.05% of the sodium deoxycholate and 0.06% of the Triton X-100. By applying the extracting solution, the yield of protein obtained from fat is higher, and compared with other extracting methods, the extracting solution is simple in formula and easy and convenient to operate, and the extracted protein solution is clear and high in purity.

Description

Technical Field

[0001] The invention belongs to the technical field of protein separation, and in particular relates to an extracting solution and an extraction method for extracting protein from adipose tissue. Background Art

[0002] Protein is an important molecule in cells and is involved in the structure and function of cells. Protein extracted from adipose tissue can be used in biological research, drug development and other fields.

[0003] Adipose tissue is composed of adipocytes, surrounded by connective tissue and extracellular matrix, and contains high levels of lipids. These components can interfere with protein extraction, especially the presence of lipids, which can co-precipitate with proteins during protein extraction, resulting in low yield and poor quality of protein samples. The location and physiological state of adipose tissue are different, the composition of adipose tissue protein is different, and it is complex, making the extraction process more complicated. Extracting total protein from cultured cells and tissues is a very common experimental procedure in the laboratory. It is relatively easy to extract total protein from most types of cells and tissues, but due to the complex composition of adipose tissue and high lipid content mentioned above, extracting total protein from adipose tissue is a challenging process.

[0004] Currently, the commonly used methods for extracting total protein from adipose tissue include:

[0005] 1) RIPA buffer extraction method: Adipose tissue is homogenized with a buffer containing a surfactant (such as RIPA buffer). After centrifugation, the liquid component under the fat layer is collected as protein. This method is relatively simple, but the protein yield is very low because the surfactant often causes the protein to form tiny particles, resulting in protein loss.

[0006] 2) Organic solvent extraction TCA precipitation method, including the following three methods:

[0007] a) Adipose tissue was centrifuged on a sucrose density gradient and proteins were precipitated by adding trichloroacetic acid (TCA) to acetone at -20°C overnight.

[0008] b) Adipose tissue was lysed with an organic solvent extraction solution (a mixture of chloroform and methanol) and precipitated with TCA as above.

[0009] c) Adipose tissue was homogenized with organic solvent extract (TEF and chloroform), the aqueous phase and the chloroform phase were separated by centrifugation, the aqueous phase and the interface were removed, and dried by centrifugal evaporation.

[0010] The common problems of the above three methods are that the operation is complicated and the yield is low. In addition, the organic solvents such as acetone and chloroform used have odor and are unsafe to use. Summary of the invention

[0011] The purpose of the present invention is to provide an extracting solution and an extraction method for extracting protein from adipose tissue, thereby making up for the deficiencies of the prior art.

[0012] The present invention first provides an extracting solution, wherein the extracting solution is a PBS buffer solution added with sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100;

[0013] As a specific record of the embodiment, the concentration of the PBS buffer is 50mM;

[0014] Preferably, the pH value of the PBS buffer is 7.0-7.4.

[0015] Furthermore, in the extract, the concentrations of sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100 are as follows: 50 mM PBS, 0.25 M sucrose, 0.5 mM EDTA, 0.5 mM SDS, 0.05% sodium deoxycholate, and 0.06% TritonX-100.

[0016] The present invention also provides a use of the extract, which is used to extract protein from adipose tissue;

[0017] In another aspect, the present invention provides a product for extracting protein from adipose tissue, wherein the product further comprises the above-mentioned extract.

[0018] In yet another aspect, the present invention provides a method for extracting protein from a fat sample, which comprises using the above-mentioned extracting solution to extract protein from adipose tissue.

[0019] Furthermore, in the method, the mass volume ratio of the extract and the fat sample is 1:10 g / ml.

[0020] Beneficial effects:

[0021] The extract of the present invention can achieve a higher protein yield from fat. Compared with other extraction methods, the extract of the present invention has a simple formula, a simple operation method, and the extracted protein solution is clear and has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :SDS-PAGE gel electrophoresis of rat adipose tissue treated in different ways,

[0023] Wherein M is a protein marker, 1 is a protein extracted by a commercially available tissue protein extraction kit (positive control), 2 is a protein extracted by this method, and 3 is a supernatant sample obtained by grinding rat adipose tissue and centrifuging it (negative control). DETAILED DESCRIPTION

[0024] The present invention is described in detail below in conjunction with embodiments and drawings.

[0025] Example 1: Screening for extracting protein from adipose tissue

[0026] By testing and optimizing rat adipose tissue as the experimental sample, the composition of the solution was determined to be PBS, sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100. PBS provides a protein buffer solution system; sucrose can improve the stability of the protein solution; EDTA is a metal chelator that prevents metal ions from interfering with proteins and ensures the purity and activity of proteins; SDS is a surfactant that can destroy the non-covalent bonds between proteins and help protein separation; sodium deoxycholate is a water-soluble anionic detergent that can effectively destroy the interaction between multiple proteins and increase protein solubility; and Triton X-100 is a non-ionic detergent that can effectively dissolve cell membranes, release proteins inside cells, and increase protein yield.

[0027] At the same time, univariate analysis was performed on each component based on a certain concentration, and the protein concentration of the extract of 0.5 g rat adipose tissue samples in different treatment groups was measured using a BCA protein assay kit to determine the optimal concentration and combination.

[0028] The specific extraction method is as follows:

[0029] 1) Weigh the fat body tissue;

[0030] 2) Then add 10 times the fat mass (g / ml) of the above protein extraction solution;

[0031] 3) Manually homogenize the tissue using a tissue pestle until no visible tissue remains; then incubate on ice for 15 min;

[0032] The incubated samples were vortexed and centrifuged at 15,000 × g for 20 min at 4°C;

[0033] 4) Use a 0.45 um water-soluble membrane to filter to obtain the soluble protein supernatant, and use the BCA protein assay kit to determine the protein concentration in the extract.

[0034] Table 1: Extraction effect table of different PBS concentrations

[0035]

[0036]

[0037] Table 2: Extraction effect of different sucrose concentrations

[0038]

[0039] Table 3: Extraction effect of different EDTA concentrations

[0040]

[0041] Table 4: Extraction effect of different SDS concentrations

[0042]

[0043] Table 5: Extraction effect of different sodium deoxycholate concentrations

[0044]

[0045]

[0046] Table 6: Extraction effect of different Triton X-100 concentrations

[0047]

[0048] Based on the test in the above table, the concentration of PBS buffer was finally determined to be 50 mM, the pH value was 7.0-7.4, and the concentrations of sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100 were as follows: 50 mM PBS, 0.25 M sucrose, 0.5 mM EDTA, 0.5 mM SDS, 0.05% sodium deoxycholate, 0.06% Triton X-100.

[0049] The prepared extract was used to extract protein from rat adipose tissue, and a commercially available tissue protein extraction kit was used as a control.

[0050] The results are as follows Figure 1 As shown, the commercially available adipose tissue extraction kit extracts a large number of protein components, and the content of each main component is clearly distinguished (the darker the color, the more the content of the corresponding molecular weight protein), while the results of directly grinding the sample without adding the extraction reagent show that the protein types are few and the content is low. The protein extracted by the extract of the present invention is consistent with the comparison result, the effect is good, and the formula is simple and the operation is more convenient.

[0051] The BCA method was further used to determine that the concentration of the protein solution extracted from 1g of rat adipose tissue by this method was 36.3mg / ml, while the concentration of the protein solution extracted after directly grinding 1g of rat adipose tissue was 1.7mg / ml. The total protein yield obtained by the method of the present invention can be relatively increased by 21 times.

[0052] In summary, the protein yield from fat can be higher by using the extract of the present invention. Compared with other extraction methods, the extract of the present invention has a simple formula, a simple operation method, and the extracted protein solution is clear and has high purity.

Claims

1. A protein extract, characterized in that: The protein extract is a PBS buffer solution added with sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100.

2. The protein extract according to claim 1, characterized in that The concentration of the PBS buffer is 50 mM.

3. The protein extract according to claim 1, characterized in that The pH value of the PBS buffer is 7.0-7.

4.

4. The protein extract according to claim 1, characterized in that The concentrations of sucrose, EDTA, SDS, sodium deoxycholate and Triton X-100 are as follows: 50 mM PBS, 0.25 M sucrose, 0.5 mM EDTA, 0.5 mM SDS, 0.05% sodium deoxycholate, 0.06% Triton X-100.

5. Use of the protein extract according to any one of claims 1 to 4 in extracting protein from adipose tissue.

6. A product for extracting protein from adipose tissue, characterized in that: The product contains the protein extract according to any one of claims 1 to 4.

7. A method for extracting protein from a fat sample, characterized in that: The method is to use the protein extract according to any one of claims 1 to 4 to extract protein from adipose tissue.

8. The method according to claim 7, characterized in that The mass volume ratio of the extract to the adipose tissue is 1:10 g / ml.

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