Rapid column type protein extraction kit and preparation method thereof

Abstract

The invention relates to the technical field of kits, and discloses a rapid column type protein extraction kit and a preparation method thereof. The rapid column type protein extraction kit comprises a lysis solution, a centrifugal tubular column, a collecting tube and a plastic grinding rod. The lysis solution is prepared from Tris, sodium chloride, EDTA-Na2, SDS and Triton X-100 according to a specific proportion, and has the characteristics of efficient lysis and protease inhibition. The lysate formula is high in universality, can adapt to various sample types, and is standardized in operation process and good in repeatability. The sample transfer loss is reduced through the column type design, and the protein yield and purity are remarkably improved by matching with the lysate. Compared with a traditional method, extraction time is shortened, and the method is especially suitable for trace samples.

Description

Technical Field

[0001] This invention relates to the field of reagent kit technology, specifically to a rapid column-based protein extraction kit and its preparation method. Background Technology

[0002] Protein extraction is a fundamental step in molecular biology research, and its efficiency and quality directly affect the reliability of downstream experimental results. Traditional protein extraction methods typically involve lysis buffers such as RIPA, NP-40, and SDS combined with repeated centrifugation. This process is not only cumbersome and time-consuming but also prone to sample loss or protease degradation, leading to decreased protein yield. For trace samples (such as small amounts of adherent cells or precious tissues), conventional methods often fail to yield sufficient protein, severely limiting the feasibility of small-sample studies. Furthermore, the formulations of lysis buffers in existing technologies lack universality, making it difficult to simultaneously meet the high-efficiency lysis requirements of different sample types (such as adherent cells, suspension cells, or rigid tissues), and incomplete removal of cell debris during extraction can easily interfere with subsequent experimental analysis. While commercial protein extraction kits have simplified the process to some extent, they still suffer from unstable lysis efficiency and complex centrifugation procedures. Therefore, developing an efficient, stable, and versatile protein extraction technology is crucial for improving the accuracy and reproducibility of protein research. Summary of the Invention

[0003] (a) Technical problems to be solved:

[0004] To address the shortcomings of existing technologies, this invention provides a rapid column-based protein extraction kit and its preparation method, which solves the problems of low protein extraction efficiency and small sample size protein extraction.

[0005] (II) Technical Solution:

[0006] A rapid column-based protein extraction kit includes: Lysis solution, centrifuge columns and collection tubes, and plastic grinding rods. The lysis buffer was prepared as follows: Tris, sodium chloride, EDTA-Na2, and SDS were added to a mixing tank in a ratio of (4-10)g:(3-8)g:(0.1-2)g:(1-6)g. Distilled water was then added, followed by Triton X-100. The mixture was stirred until dissolved, and the pH was adjusted to 5.2-6.9 with concentrated hydrochloric acid. Lysis solution.

[0007] Preferably, the method for extracting proteins from adherent cells using the kit is as follows: Wash adherent cells with pre-cooled phosphate buffer in a culture dish, remove the supernatant, and add... Lysis buffer was used, and the lysed cells were pipetted and then transferred to a pre-cooled centrifuge column sleeve. The cells were centrifuged, and the protein solution in the collection tube was retained and transferred to a clean centrifuge tube to obtain the protein solution of adherent cells.

[0008] Preferably, in the method for extracting adherent cell proteins using the kit, the centrifugation conditions are 12000-15000×g for 30s.

[0009] Preferably, the method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add pre-cooled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge to wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add... Lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-cooled centrifuge column sleeve. After centrifugation, the collection tube was removed and placed on ice. The centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a protein solution of suspended cells.

[0010] Preferably, in the method for extracting suspended cell proteins using the kit, the centrifugation conditions for the first centrifugation are 1000×g for 2-3 min; and the centrifugation conditions for the second centrifugation are 12000-15000×g for 30 s.

[0011] Preferably, in the method for extracting suspended cell proteins using the kit, the phosphate buffer is prepared as follows: NaCl, potassium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride are added to deionized water in a ratio of (5-10)g:(0.15-0.55)g:(1-2)g:(0.1-0.5)g, stirred to dissolve, filtered to remove bacteria, and the filtrate is phosphate buffer.

[0012] Preferably, the method for extracting proteins from tissue samples using the kit is as follows: Add the tissue sample to a pre-chilled centrifuge tube, grind it using a plastic grinder, and add... The lysis buffer was further ground, then incubated at room temperature, centrifuged, and the protein solution was collected and transferred to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0013] Preferably, in the method for extracting proteins from tissue samples using the kit, the incubation time at room temperature is 1-2 min, and the centrifugation conditions are 12000-15000×g for 1-2 min.

[0014] (III) Beneficial Technical Effects:

[0015] This invention provides a highly efficient and convenient rapid column-based protein extraction kit, which significantly improves protein extraction efficiency and quality by optimizing the lysis buffer formulation and column centrifugation technology. The lysis buffer uses a specific ratio of Tris, sodium chloride, EDTA-Na2, and SDS, adjusted with Triton X-100. Its pH range is adaptable to various sample types, effectively inhibiting protease activity and preventing protein degradation while gently lysing cell membranes. Compared to traditional RIPA lysis buffers, the buffer used in this invention... The extraction time of the lysis buffer is shortened by more than 30%, and the protein yield is increased by 20%-25%, making it especially suitable for micro-samples.

[0016] This invention achieves rapid separation and impurity removal of protein solutions by introducing pre-cooled centrifuge columns and collection tubes. The column design avoids sample loss caused by repeated transfers in traditional centrifuge tubes, and the precise optimization of centrifugation force and time ensures efficient protein enrichment while reducing cell debris residue.

[0017] This invention solves the problems of low efficiency and poor adaptability to small samples in traditional protein extraction methods by integrating high-efficiency lysis buffer and column separation technology. Detailed Implementation

[0018] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0019] Example 1: A method for preparing a rapid column-based protein extraction kit is as follows:

[0020] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to the culture dish to wash the adherent cells, aspirate the supernatant, and add 300 μL of... Lysis buffer was used, and the lysed cells were pipetted and transferred to a pre-cooled centrifuge column sleeve. The cells were centrifuged at 12000×g for 30 seconds. The protein solution in the collection tube was retained and transferred to a clean centrifuge tube to obtain the protein solution of adherent cells. The lysis buffer was prepared as follows: Add 4g Tris, 3g sodium chloride, 0.1g EDTA-Na2, and 1g SDS to a mixing tank, then add 800mL of distilled water and 5mL of Triton X-100. Stir to dissolve, and adjust the pH to 6.9 with concentrated hydrochloric acid. Lysis solution.

[0021] S2. The method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 3 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add 300 μL of [unspecified ingredient]. Lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-chilled centrifuge column sleeve and centrifuged at 12000×g for 30 seconds. The collection tube was placed on ice, the centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a suspended protein solution of cells. The phosphate buffer was prepared as follows: 5g NaCl, 0.15g potassium dihydrogen phosphate, 1g disodium hydrogen phosphate, and 0.1g potassium chloride were added to 100mL of deionized water. The mixture was stirred to dissolve, filtered sterilely, and the filtrate was the phosphate buffer.

[0022] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-chilled centrifuge tube, grind using a plastic grinder, and add 300 μL of [unspecified ingredient]. The lysis buffer was further ground, then incubated at room temperature for 1 min, centrifuged at 15000×g for 1 min, and the protein solution was collected and transferred to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0023] Example 2: A method for preparing a rapid column-based protein extraction kit is as follows:

[0024] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to the culture dish to wash the adherent cells, aspirate the supernatant, and add 300 μL of... Lysis buffer was used, and the lysed cells were pipetted and transferred to a pre-cooled centrifuge column sleeve. The cells were centrifuged at 15000×g for 30 seconds. The protein solution in the collection tube was retained and transferred to a clean centrifuge tube to obtain the protein solution of adherent cells. The lysis buffer was prepared as follows: Add 10g Tris, 8g sodium chloride, 2g EDTA-Na2, and 6g SDS to a mixing tank, then add 800mL of distilled water and 3mL of Triton X-100. Stir to dissolve, and adjust the pH to 6.0 with concentrated hydrochloric acid. Lysis solution.

[0025] S2. The method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 2 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add 300 μL of [unspecified ingredient]. Lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-chilled centrifuge column sleeve and centrifuged at 15000×g for 30 seconds. The collection tube was then placed on ice, the centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a suspended protein solution of cells. The phosphate buffer was prepared as follows: 10g NaCl, 0.55g potassium dihydrogen phosphate, 2g disodium hydrogen phosphate, and 0.5g potassium chloride were added to 100mL of deionized water. The mixture was stirred to dissolve, filtered sterilely, and the filtrate was the phosphate buffer.

[0026] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-chilled centrifuge tube, grind using a plastic grinder, and add 300 μL of [unspecified ingredient]. The lysis buffer was further ground, then incubated at room temperature for 2 minutes, centrifuged at 15000×g for 2 minutes, and the protein solution was collected and transferred to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0027] Example 3: A method for preparing a rapid column-based protein extraction kit is as follows:

[0028] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to the culture dish to wash the adherent cells, aspirate the supernatant, and add 300 μL of... Lysis buffer was used, and the lysed cells were pipetted and transferred to a pre-cooled centrifuge column sleeve. The cells were centrifuged at 13000×g for 30 seconds. The protein solution in the collection tube was retained and transferred to a clean centrifuge tube to obtain the protein solution of adherent cells. The lysis buffer was prepared as follows: Add 7g Tris, 5.5g sodium chloride, 1.2g EDTA-Na2, and 3.5g SDS to a mixing tank, then add 800mL of distilled water and 2mL of Triton X-100. Stir to dissolve, and adjust the pH to 5.2 with concentrated hydrochloric acid. Lysis solution.

[0029] S2. The method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 2 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add 300 μL of [unspecified ingredient]. Lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-chilled centrifuge column sleeve and centrifuged at 13000×g for 30 seconds. The collection tube was then placed on ice, the centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a protein solution of suspended cells. The phosphate buffer was prepared as follows: 7g NaCl, 0.35g potassium dihydrogen phosphate, 1.5g disodium hydrogen phosphate, and 0.3g potassium chloride were added to 100mL of deionized water. The mixture was stirred to dissolve, filtered sterilely, and the filtrate was the phosphate buffer.

[0030] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-chilled centrifuge tube, grind using a plastic grinder, and add 300 μL of [unspecified ingredient]. The lysis buffer was further ground, then incubated at room temperature for 2 minutes, centrifuged at 13000×g for 2 minutes, and the protein solution was collected and transferred to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0031] Example 4: A method for preparing a rapid column-based protein extraction kit is as follows:

[0032] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to the culture dish to wash the adherent cells, aspirate the supernatant, and add 300 μL of... Lysis buffer was used, and the lysed cells were pipetted and transferred to a pre-cooled centrifuge column sleeve. The cells were centrifuged at 15000×g for 30 seconds. The protein solution in the collection tube was retained and transferred to a clean centrifuge tube to obtain the protein solution of adherent cells. The lysis buffer was prepared as follows: Add 7g Tris, 8g sodium chloride, 2g EDTA-Na2, and 4g SDS to a mixing tank, then add 800mL of distilled water and 2mL of Triton X-100. Stir to dissolve, and adjust the pH to 6.9 with concentrated hydrochloric acid. Lysis solution.

[0033] S2. The method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 3 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add 300 μL of [unspecified ingredient]. Lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-chilled centrifuge column sleeve and centrifuged at 15000×g for 30 seconds. The collection tube was placed on ice, the centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a suspended protein solution. The phosphate buffer was prepared as follows: 6g NaCl, 0.15g potassium dihydrogen phosphate, 1g disodium hydrogen phosphate, and 0.3g potassium chloride were added to 100mL of deionized water. The mixture was stirred to dissolve, filtered sterilely, and the filtrate was the phosphate buffer.

[0034] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-chilled centrifuge tube, grind using a plastic grinder, and add 300 μL of [unspecified ingredient]. The lysate was further ground, then incubated at room temperature for 1 min, centrifuged at 12000×g for 2 min, and the protein solution was collected and transferred to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0035] Comparative Example 1: Using RIPA lysis buffer instead of the solution in Example 1 Lysis solution.

[0036] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to a culture dish to wash the adherent cells, aspirate the supernatant, add 300 μL of RIPA lysis buffer, pipette and regurgitate, then transfer the lysed cells to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 s, retain the protein solution in the collection tube, and transfer it to a clean centrifuge tube to obtain the protein solution of the adherent cells. The RIPA lysis buffer (catalog number: R301900-100 mL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0037] S2. The method for extracting protein from suspended cells using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 3 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, then add 300 μL of RIPA lysis buffer, vortex to lyse the cells for 15 seconds, transfer to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 seconds, remove, place the collection tube on ice, discard the centrifuge column, and transfer the protein solution in the collection tube to a clean centrifuge tube to obtain the protein solution of the suspended cells. The phosphate buffer is prepared as follows: Add 5 g NaCl, 0.15 g potassium dihydrogen phosphate, 1 g disodium hydrogen phosphate, and 0.1 g potassium chloride to 100 mL of deionized water, stir to dissolve, filter to sterilize, and the filtrate is phosphate buffer.

[0038] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-cooled centrifuge tube, grind with a plastic grinder, add 300 μL of RIPA lysis buffer, continue grinding, then incubate at room temperature for 1 min, centrifuge at 15000×g for 1 min, collect the protein solution and transfer it to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0039] Comparative Example 2: Using NP-40 lysis buffer instead of the solution in Example 1 Lysis solution.

[0040] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to a culture dish to wash the adherent cells, aspirate the supernatant, add 300 μL of NP-40 lysis buffer, pipette and regurgitate, then transfer the lysed cells to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 s, retain the protein solution in the collection tube, and transfer it to a clean centrifuge tube to obtain the protein solution of the adherent cells. The NP-40 lysis buffer (catalog number: N748603-100 mL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0041] S2. The method for extracting protein from suspended cells using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 3 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, then add 300 μL of RIPA lysis buffer, vortex to lyse the cells for 15 seconds, transfer to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 seconds, remove, place the collection tube on ice, discard the centrifuge column, and transfer the protein solution in the collection tube to a clean centrifuge tube to obtain the protein solution of the suspended cells. The phosphate buffer is prepared as follows: Add 5 g NaCl, 0.15 g potassium dihydrogen phosphate, 1 g disodium hydrogen phosphate, and 0.1 g potassium chloride to 100 mL of deionized water, stir to dissolve, filter to sterilize, and the filtrate is phosphate buffer.

[0042] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-cooled centrifuge tube, grind with a plastic grinder, add 300 μL of NP-40 lysis buffer, continue grinding, then incubate at room temperature for 1 min, centrifuge at 15000×g for 1 min, collect the protein solution and transfer it to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0043] Comparative Example 3: Using SDS lysis buffer instead of the solution in Example 1 Lysis solution.

[0044] S1. The method for extracting proteins from adherent cells using the kit is as follows: Add 5 mL of pre-chilled phosphate buffer to a culture dish to wash the adherent cells, aspirate the supernatant, add 300 μL of SDS lysis buffer, pipette and regurgitate, then transfer the lysed cells to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 s, retain the protein solution in the collection tube, and transfer it to a clean centrifuge tube to obtain the protein solution of the adherent cells. The SDS lysis buffer (catalog number: S750312-100 mL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0045] S2. The method for extracting protein from suspended cells using the kit is as follows: Centrifuge the suspended cells at low speed, add 5 mL of pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge at 1000×g for 3 minutes, wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, then add 300 μL of SDS lysis buffer, vortex to lyse the cells for 15 seconds, transfer to a pre-chilled centrifuge column sleeve, centrifuge at 12000×g for 30 seconds, remove, place the collection tube on ice, discard the centrifuge column, and transfer the protein solution in the collection tube to a clean centrifuge tube to obtain the protein solution of the suspended cells. The phosphate buffer is prepared as follows: Add 5 g NaCl, 0.15 g potassium dihydrogen phosphate, 1 g disodium hydrogen phosphate, and 0.1 g potassium chloride to 100 mL of deionized water, stir to dissolve, filter to sterilize, and the filtrate is phosphate buffer.

[0046] S3. The method for extracting protein from tissue samples using the kit is as follows: Add 25 mg of tissue sample to a pre-cooled centrifuge tube, grind with a plastic grinding rod, add 300 μL of SDS lysis buffer, continue grinding, then incubate at room temperature for 1 min, centrifuge at 15000×g for 1 min, collect the protein solution and transfer it to a clean centrifuge tube to obtain the protein solution of the tissue sample.

[0047] Table 1. Time and system for protein extraction using the kit.

[0048]

[0049]

[0050] Example 1: Through optimized formulation The lysis buffer (pH 6.9, containing Tris, SDS, Triton X-100, etc.) combined with a pre-cooled centrifuge column and disposable plastic grinding rods enables efficient and rapid protein extraction. Tissue sample protein extraction takes only 300 seconds, with a protein yield of 21.5 μg, significantly superior to traditional methods. This approach also avoids sample loss and is suitable for micro-sample and high-throughput experiments, combining high efficiency, stability, and convenience.

[0051] Comparative Example 1 used traditional RIPA lysis buffer, which had weak lysis efficiency and protease inhibition ability, resulting in an extended extraction time of adherent cells to 420 seconds and a protein yield of 20.2 μg. This indicates that it is not efficient enough when processing small amounts of samples and cannot meet the requirements for rapid extraction.

[0052] Comparative Example 2 used NP-40 lysis buffer, but its core problems lie in insufficient lysis strength and narrow applicability. While NP-40, as a non-ionic detergent, can preserve the native conformation of proteins, its lysis efficiency for membrane proteins or tissue samples is low. The table data shows that the yield of suspended cell proteins was only 20.3 μg, and the extraction time was as long as 430 seconds, far inferior to Example 1. This is because NP-40 cannot effectively break down cell walls or dissolve inclusion body proteins, resulting in some target proteins remaining in the precipitate. Furthermore, NP-40 lacks strong denaturing agents (such as SDS), making it difficult to completely inhibit protease activity, especially in tissue sample processing where it easily induces protein degradation. Although its yield for adherent cells (21.1 μg) is close to that of Example 1, the extraction time (540 seconds) is significantly increased, indicating that it is more suitable for soluble protein studies and cannot meet the high-efficiency extraction requirements of complex samples.

[0053] Comparative Example 3 used SDS lysis buffer. Although the protein yield (22.4 μg) was slightly higher than that of Example 1, its excessive denaturation and degradation of heat-sensitive proteins were significant problems. While the strong denaturing effect of SDS can completely dissolve poorly soluble proteins, it also destroys the native protein structure, limiting downstream applications (such as Co-IP or enzyme activity assays). The tissue sample extraction time in the table (350 seconds) was shorter than RIPA and NP-40, but still longer than that of Example 1 (300 seconds). Furthermore, SDS may cause irreversible denaturation of heat-sensitive proteins (such as phosphorylated proteins) under high temperature or prolonged treatment. In addition, residual SDS can interfere with mass spectrometry analysis, requiring additional removal steps and increasing experimental complexity. Therefore, although SDS performs reasonably well in terms of yield, its destructive lysis and poor compatibility limit its application in the study of multifunctional proteins.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a rapid column-based protein extraction kit, characterized in that, The rapid column-based protein extraction kit includes: Pr lysis buffer, centrifuge column and collection tube, plastic grinding rod; the aforementioned The preparation method of Pr lysis buffer is as follows: Add Tris, sodium chloride, EDTA-Na2, and SDS in a ratio of (4-10)g:(3-8)g:(0.1-2)g:(1-6)g to a mixing tank, then add distilled water, add Triton X-100, stir to dissolve, and adjust the pH to 5.2-6.9 with concentrated hydrochloric acid to obtain the solution. Pr lysis buffer.

2. The method for preparing the rapid column-based protein extraction kit according to claim 1, characterized in that, The method for extracting adherent cell proteins using the kit is as follows: Add pre-cooled phosphate buffer to the culture dish to wash the adherent cells, aspirate the supernatant, and add... Pr lysis buffer, pipette and then transfer the lysed cells to a pre-cooled centrifuge column sleeve, centrifuge, retain the protein solution in the collection tube and transfer it to a clean centrifuge tube to obtain the protein solution of adherent cells.

3. The method for preparing the rapid column-based protein extraction kit according to claim 1, characterized in that, The method for extracting suspended cell proteins using the kit is as follows: Centrifuge the suspended cells at low speed, add pre-chilled phosphate buffer to the centrifuge tube, vortex for 5 seconds, centrifuge to wash the cells, aspirate the supernatant, retain the same volume of phosphate buffer as the cell pellet, vortex to resuspend the cells, and then add... Pr lysis buffer was used to lyse cells by vortexing for 15 seconds. The lysate was then transferred to a pre-cooled centrifuge column sleeve. After centrifugation, the collection tube was removed and placed on ice. The centrifuge column was discarded, and the protein solution in the collection tube was transferred to a clean centrifuge tube to obtain a protein solution of suspended cells.

4. The method for preparing the rapid column-based protein extraction kit according to claim 1, characterized in that, The method for extracting proteins from tissue samples using the kit is as follows: Add the tissue sample to a pre-chilled centrifuge tube, grind it using a plastic grinding stick, and add... Pr lysis buffer, continue grinding, then incubate at room temperature, centrifuge, collect the protein solution and transfer it to a clean centrifuge tube to obtain the protein solution of the tissue sample.

5. The method for preparing the rapid column-based protein extraction kit according to claim 2, characterized in that, In the method for extracting adherent cell proteins using the kit, the phosphate buffer is prepared as follows: Add NaCl, potassium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride in a ratio of (5-10)g:(0.15-0.55)g:(1-2)g:(0.1-0.5)g to deionized water, stir to dissolve, filter to remove bacteria, and the filtrate is phosphate buffer.

6. The method for preparing the rapid column-based protein extraction kit according to claim 2, characterized in that, In the method for extracting adherent cell proteins using the kit, the centrifugation conditions are 12000-15000×g for 30s.

7. The method for preparing the rapid column-based protein extraction kit according to claim 3, characterized in that, In the method for extracting suspended cell proteins using the kit, the first centrifugation is performed at 1000×g for 2-3 min; the second centrifugation is performed at 12000-15000×g for 30 s.

8. The method for preparing the rapid column-based protein extraction kit according to claim 4, characterized in that, In the method for extracting proteins from tissue samples using the kit, the incubation time at room temperature is 1-2 min, and the centrifugation conditions are 12000-15000×g for 1-2 min.

9. A rapid column-based protein extraction kit obtained by the preparation method according to any one of claims 1-8.

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