Application of extracting protein by using novel adsorption material

Abstract

The invention discloses an application of extracting protein by using a novel adsorption material, which comprises a protein extraction method capable of extracting protein from animal tissues, plant tissues, cell lines, serum or plasma.

Description

Technical Field

[0001] The present invention relates to the technical field of application of extracting protein by using a novel adsorption material, and in particular to an application of extracting protein by using a novel adsorption material. Background Art

[0002] Currently, ion exchange is a commonly used protein purification technique, separating proteins based on the surface charge characteristics of protein molecules. Proteins carry different net charges at different pH values, allowing them to undergo electrostatic interactions with ion exchangers. Ion exchangers are divided into cation exchangers and anion exchangers, which are used to bind positively and negatively charged proteins, respectively. However, they also have some disadvantages: they are sensitive to experimental conditions, may cause protein denaturation, and have low charge separation efficiency in complex samples. Subsequent methods such as gel filtration chromatography and affinity chromatography are required to achieve higher purity requirements. Summary of the Invention

[0003] In order to solve certain technical problems existing in the prior art, the purpose of this application is to provide an application for extracting protein using a new adsorption material, which simplifies the operation process of the traditional extraction method, shortens the protein extraction time, increases the total amount and purity of protein extraction, and is beneficial to downstream applications.

[0004] In order to solve the above existing technical problems, one of the objectives of this application is achieved by adopting the following technical solutions:

[0005] The invention discloses an application of extracting protein by using a novel adsorption material, which includes a protein extraction method. The method can extract protein from animal tissue, plant tissue, cell line, serum or plasma.

[0006] Furthermore, when extracting protein from animal tissue, the following steps are included:

[0007] S1: Mince fresh or frozen animal tissue, add lysis buffer containing protease inhibitors, homogenize on ice or use ultrasonic disruption to destroy cell membranes, and incubate on ice for a certain period of time, usually 30 minutes. Vortex and shake at intervals to ensure sufficient lysis.

[0008] S2 removes unlysed cell debris and other insoluble substances (such as cell debris, membrane lipids, etc.) by high-speed centrifugation (e.g., 12000 rpm, 10-20 minutes at 4°C), and collects the supernatant as the crude protein solution;

[0009] S3 is adsorbed by adding macroporous adsorption resin, and the resin and its adsorbent are fully shaken and centrifuged to remove the retained liquid;

[0010] The liquid obtained in S4 is the protein sample.

[0011] Furthermore, when extracting protein from plant tissue, the following steps are included:

[0012] S1. Plant material is usually ground into a fine powder using liquid nitrogen to disrupt the cell walls. Extraction buffer containing a polymerase inhibitor and a reducing agent is added and mixed thoroughly. Cellulose and other impurities are removed by filtration or centrifugation. The reducing agent is DTT.

[0013] S2, adding macroporous adsorption resin for adsorption, fully shaking and centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0014] S3. The obtained liquid is the protein sample.

[0015] Furthermore, when extracting protein from a cell line, the following steps are included:

[0016] S1. Collect cells and add lysis buffer. Vortex to help cells completely break and release proteins.

[0017] S2. Remove unlysed cell debris and other insoluble substances by high-speed centrifugation, and collect the supernatant as the crude protein solution. The high-speed centrifugation state is 12000 rpm, 4°C for 10-20 minutes;

[0018] S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0019] S4. The obtained liquid is the protein sample.

[0020] Furthermore, when extracting protein from plasma or serum, the following steps are included:

[0021] S1, salt and other small molecules can be removed by dialysis or ultrafiltration;

[0022] S2. Protein can be selectively concentrated using ammonium sulfate precipitation;

[0023] S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0024] S4. The obtained liquid is the protein sample.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The method of the present application uses macroporous adsorption resin to adsorb various impurities in the sample, including but not limited to nucleic acids, lipids, ionic small molecules, metabolites, etc., but does not adsorb proteins. The resin bound to the impurities is separated from the sample by centrifugation, and the resulting clear solution is the protein sample, which can be directly used in downstream experiments. This simplifies the operation process of traditional extraction methods, shortens protein extraction time, increases the total amount and purity of protein extraction, and is beneficial for downstream applications. DETAILED DESCRIPTION

[0027] Below, the present application is further described in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0028] In the description of this application, it should be understood that the terms "up", "down", "left", "right", etc., which indicate directions or positional relationships, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operated in a specific direction. Therefore, it should not be understood as a limitation on this application.

[0029] The terms "first", "second", etc. in this application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those described here, and the objects distinguished by "first", "second", etc. are generally of one type, and the number of objects is not limited. For example, the first object can be one or more. In addition, "and / or" in the specification and claims represents at least one of the connected objects, and the character " / " generally indicates that the objects before and after are an "or" relationship of an application of extracting proteins using a new adsorbent material.

[0030] Example 1:

[0031] An application of protein extraction using a novel adsorption material, comprising a protein extraction method capable of extracting protein from animal tissue, plant tissue, cell lines, serum, or plasma. The method of the present application uses a macroporous adsorption resin to adsorb various impurities in the sample, including but not limited to nucleic acids, lipids, ionic small molecules, metabolites, etc., without adsorbing protein. The resin bound to the impurities is separated from the sample by centrifugation, and the resulting clear protein is the protein sample, which can be directly used in downstream experiments. This simplifies the operating procedures of traditional extraction methods, shortens protein extraction time, increases the total amount and purity of protein extraction, and is beneficial for downstream applications.

[0032] Furthermore, when extracting protein from animal tissue, the following steps are included:

[0033] S1: Mince fresh or frozen animal tissue, add lysis buffer containing protease inhibitors, homogenize on ice or use ultrasonic disruption to destroy cell membranes, and incubate on ice for a certain period of time, usually 30 minutes. Vortex and shake at intervals to ensure sufficient lysis.

[0034] S2 removes unlysed cell debris and other insoluble substances (such as cell debris, membrane lipids, etc.) by high-speed centrifugation (e.g., 12000 rpm, 10-20 minutes at 4°C), and collects the supernatant as the crude protein solution;

[0035] S3 is adsorbed by adding macroporous adsorption resin, and the resin and its adsorbent are fully shaken and centrifuged to remove the retained liquid;

[0036] The liquid obtained in S4 is the protein sample.

[0037] Furthermore, when extracting protein from plant tissue, the following steps are included:

[0038] S1. Plant material is usually ground into a fine powder using liquid nitrogen to disrupt the cell walls. Extraction buffer containing a polymerase inhibitor and a reducing agent is added and mixed thoroughly. Cellulose and other impurities are removed by filtration or centrifugation. The reducing agent is DTT.

[0039] S2, adding macroporous adsorption resin for adsorption, fully shaking and centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0040] S3. The obtained liquid is the protein sample.

[0041] Furthermore, when extracting proteins from cell lines, the following steps are included:

[0042] S1. Collect cells and add lysis buffer. Vortex to help cells completely break and release proteins.

[0043] S2. Remove unlysed cell debris and other insoluble substances by high-speed centrifugation, and collect the supernatant as the crude protein solution. The high-speed centrifugation state is 12000 rpm, 4°C for 10-20 minutes;

[0044] S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0045] S4. The obtained liquid is the protein sample.

[0046] Furthermore, when extracting proteins from plasma or serum, the following steps are included:

[0047] S1, salt and other small molecules can be removed by dialysis or ultrafiltration;

[0048] S2. Protein can be selectively concentrated using ammonium sulfate precipitation;

[0049] S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid;

[0050] S4. The obtained liquid is the protein sample. The above embodiments are only preferred embodiments of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by those skilled in the art based on the present application shall fall within the scope of protection claimed in the present application.

Claims

1. An application of extracting protein using a new adsorption material, characterized in that: This includes protein extraction methods that can be extracted from animal tissues, plant tissues, cell lines, serum or plasma.

2. The use of a novel adsorption material for extracting protein according to claim 1, characterized in that: When extracting protein from animal tissue, the following steps are involved: S1: Mince fresh or frozen animal tissue, add lysis buffer containing protease inhibitors, homogenize on ice or use ultrasonic disruption to destroy cell membranes, and incubate on ice for a certain period of time, usually 30 minutes. Vortex and shake at intervals to ensure sufficient lysis. S2 removes unlysed cell debris and other insoluble substances (such as cell debris, membrane lipids, etc.) by high-speed centrifugation (e.g., 12000 rpm, 10-20 minutes at 4°C), and collects the supernatant as the crude protein solution; S3 is adsorbed by adding macroporous adsorption resin, and the resin and its adsorbent are fully shaken and centrifuged to remove the retained liquid; The liquid obtained in S4 is the protein sample.

3. The use of a novel adsorption material for extracting protein according to claim 2, characterized in that: When extracting protein from plant tissue, the following steps are involved: S1. Plant material is usually ground into a fine powder using liquid nitrogen to disrupt the cell walls. Extraction buffer containing a polymerase inhibitor and a reducing agent is added and mixed thoroughly. Cellulose and other impurities are removed by filtration or centrifugation. The reducing agent is DTT. S2, adding macroporous adsorption resin for adsorption, fully shaking and centrifuging to remove the resin and its adsorbate, and retaining the liquid; S3. The obtained liquid is the protein sample.

4. The use of a novel adsorption material for extracting protein according to claim 3, characterized in that: When extracting protein from a cell line, the following steps are involved: S1. Collect cells and add lysis buffer. Vortex to help cells completely break and release proteins. S2. Remove unlysed cell debris and other insoluble substances by high-speed centrifugation, and collect the supernatant as the crude protein solution. The high-speed centrifugation state is 12000 rpm, 4°C for 10-20 minutes; S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid; S4. The obtained liquid is the protein sample.

5. The use of a novel adsorption material for extracting protein according to claim 4, characterized in that: When protein is extracted from plasma or serum, the following steps are involved: S1, salt and other small molecules can be removed by dialysis or ultrafiltration; S2. Protein can be selectively concentrated using ammonium sulfate precipitation; S3, adding macroporous adsorption resin for adsorption, shaking thoroughly, centrifuging to remove the resin and its adsorbate, and retaining the liquid; S4. The obtained liquid is the protein sample.