A method for identifying small molecule and protein interactions

Abstract

The application relates to the technical field of new material application, and discloses a method for identifying small molecule and protein interaction, wherein a chemical-cleavable hydrazine magnetic bead, i.e. a hydrazine magnetic bead material containing a disulfide bond, is synthesized, the magnetic bead material is subjected to an amide chemistry-based covalent connection reaction with a small molecule, and a chemical-cleavable small molecule magnetic bead is prepared. The small molecule magnetic bead is incubated with a non-denatured protein mixture, and is subjected to elution under the action of a reducing agent and an auxiliary elution reagent, and then is identified through mass spectrometry to determine the acceptor protein interacting with the small molecule. The disulfide bond has the chemical-cleavable characteristic in the method, so that the small molecule and the protein acceptor thereof can be quickly released and only released, which not only facilitates subsequent identification but also avoids the occurrence of non-specific binding. In addition, the chemical-cleavable hydrazine magnetic bead is convenient for fixing the magnetic bead, so that the removal of excess reagents in the reaction is facilitated, and the quick synthesis of thousands of small molecule magnetic beads can be realized.

Description

Technical Field

[0001] This invention relates to the technical field of new material applications, and in particular to a method for identifying interactions between small molecules and proteins. Background Technology

[0002] Small molecule metabolites are the core of life activities. They are not only intermediate products of metabolic processes, but also regulate complex cell signaling pathways through direct or indirect interactions with proteins.

[0003] Among related technologies, there are methods for identifying specific small molecule targets, but most of them are concentrated on individual small molecules and may involve a large number of non-specific bindings. Summary of the Invention

[0004] This application provides a method for identifying interactions between small molecules and proteins. The method involves synthesizing chemically cleavable hydrazine magnetic beads, specifically hydrazine magnetic beads containing disulfide bonds. The magnetic beads are then covalently linked to small molecules using amide chemistry to prepare chemically cleavable small molecule magnetic beads. The small molecule magnetic beads are incubated with a non-denatured protein mixture and eluted under the action of a reducing agent. Subsequently, mass spectrometry is used to identify the receptor protein interacting with the small molecule. The disulfide bonds in this method are chemically cleavable, allowing for rapid release of only the small molecule and its protein receptor, facilitating subsequent identification and avoiding non-specific binding. Furthermore, the chemically cleavable hydrazine magnetic beads facilitate immobilization, simplifying the removal of excess reagents and enabling the rapid synthesis of thousands of small molecule magnetic beads.

[0005] In a first aspect, the present invention provides a method for identifying interactions between small molecules and proteins, comprising:

[0006] Carboxylated magnetic beads, aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and aqueous solution of 3-(2-dithiopyridyl)propionic hydrazide were subjected to a first water bath heating, and the first supernatant was discarded to obtain carboxylated magnetic beads after the first water bath heating.

[0007] The carboxyl magnetic beads and dithiothreitol that have undergone the first water bath heating are subjected to a second water bath heating to obtain carboxyl magnetic beads that have undergone the second water bath heating.

[0008] After the carboxyl magnetic beads were first washed after the second water bath heating, 3-(2-dithiopyridyl)propionic acid hydrazide was added and shaken to obtain chemically cleavable hydrazide magnetic beads.

[0009] After a second cleaning of the chemically cleavable hydrazide magnetic beads, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and small molecules are added, and a third water bath heating is performed to obtain chemically cleavable small molecule magnetic beads.

[0010] After a third washing of the chemically cleavable small molecule magnetic beads, a protein mixture is added and incubated.

[0011] After incubation, the second supernatant is removed to obtain incubated magnetic beads; after a fourth wash of the incubated magnetic beads, a reducing agent and an auxiliary elution reagent are added, followed by a fourth water bath heating to elute interacting proteins, and the third supernatant is collected.

[0012] The third supernatant was used as the eluent for mass spectrometry identification.

[0013] In some embodiments, the step of performing mass spectrometry identification includes: adding chloroacetamide to the eluent to react and obtain a reactant to block disulfide bonds; adding NH4HCO3 and trypsin to the reactant for enzymatic digestion to obtain an enzyme-digested sample; and performing mass spectrometry identification on the enzyme-digested sample after desalting.

[0014] In some embodiments, the reducing agent is dithiothreitol; the auxiliary elution reagent is urea.

[0015] In some embodiments, the concentration of dithiothreitol as the reducing agent is 10-100 mM, and the concentration of urea as the auxiliary elution reagent is 8 M.

[0016] In some embodiments, the heating temperature of the fourth water bath heating is 25-37°C, and the heating time is 0.5-2 hours.

[0017] In some embodiments, the protein mixture is a protein mixture dissolved in phosphate buffer.

[0018] In some embodiments, the small molecule includes a small molecule containing a carboxyl group, a carbonyl group, or a phosphate group.

[0019] In some embodiments, the small molecule includes myristic acid.

[0020] In some embodiments, the first, second, third, and fourth cleaning processes all involve rinsing with ultrapure water three times.

[0021] In some embodiments, prior to the step of heating the carboxyl magnetic beads, the aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the aqueous solution of 3-(2-dithiopyridyl)propionic acid hydrazide in a water bath, the method further includes:

[0022] Take the carboxyl magnetic bead product, shake the carboxyl magnetic bead product well, and then use a pipette to remove the carboxyl magnetic beads to remove the fourth supernatant.

[0023] This invention provides a method for identifying interactions between small molecules and proteins. The method involves synthesizing chemically cleavable hydrazide magnetic beads, specifically hydrazide magnetic beads containing disulfide bonds. The magnetic beads are then covalently linked to small molecules using amide chemistry to prepare chemically cleavable small molecule magnetic beads. The small molecule magnetic beads are incubated with a non-denatured protein mixture and eluted using a reducing agent and an elution aid. Mass spectrometry is then used to identify the receptor protein interacting with the small molecule. The chemically cleavable nature of the disulfide bonds in this method allows for rapid release of only the small molecule and its protein receptor, facilitating subsequent identification and avoiding non-specific binding. Furthermore, the chemically cleavable hydrazide magnetic beads facilitate immobilization, simplifying the removal of excess reagents and enabling the rapid synthesis of thousands of small molecule magnetic beads. Attached Figure Description

[0024] Figure 1 An exemplary flowchart illustrates a synthetic route for chemically cleavable small molecule magnetic beads according to some embodiments;

[0025] Figure 2 An exemplary flowchart is shown for identifying small molecule and protein interactions according to some embodiments;

[0026] Figure 3 A schematic diagram of the Coomassie brilliant blue staining results of Examples 1-6 provided according to some embodiments is shown as an example. Detailed Implementation

[0027] To better understand the above technical solutions, the technical solutions of this application will be described in detail below through specific implementation methods.

[0028] To address the aforementioned technical problems, this application provides a method for identifying interactions between small molecules and proteins. This method involves synthesizing chemically cleavable hydrazine magnetic beads, specifically hydrazine magnetic beads containing disulfide bonds. The magnetic beads are then covalently linked to small molecules using amide chemistry to prepare chemically cleavable small molecule magnetic beads. The small molecule magnetic beads are incubated with a non-denatured protein mixture and eluted using a reducing agent and an auxiliary elution reagent. Subsequently, mass spectrometry is used to identify the receptor protein interacting with the small molecule. The disulfide bonds in this method possess chemically cleavable properties, allowing for rapid release of only the small molecule and its protein receptor, facilitating subsequent identification and avoiding non-specific binding. Furthermore, the chemically cleavable hydrazine magnetic beads facilitate immobilization, simplifying the removal of excess reagents and enabling the rapid synthesis of thousands of small molecule magnetic beads.

[0029] Figure 1An exemplary flowchart of a chemically cleavable small molecule magnetic bead synthesis route is shown according to some embodiments. Figure 2 An exemplary flowchart is shown of a method for identifying small molecule and protein interactions according to some embodiments.

[0030] like Figure 1 As shown, carboxyl magnetic beads, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (EDC) and 3-(2-dithiopyridyl)propionic acid hydrazide aqueous solution (PDPH) were subjected to a first water bath heating, and the first supernatant was discarded to obtain carboxyl magnetic beads (i.e., pyridine dithiomethyl magnetic beads) after the first water bath heating.

[0031] In one example, the volume of the EDC aqueous solution is 50 μL and the concentration is 100 mM; the volume of the PDPH aqueous solution is 50 μL and the concentration is 100 mM.

[0032] In some embodiments, the heating temperature of the first water bath heating is 4-37°C, and the heating time is 0.5-2 hours.

[0033] In some embodiments, the initial water bath heating temperature is 37°C, and the heating time is 1 hour.

[0034] In some embodiments, before the step of heating the carboxyl magnetic beads, the aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and the aqueous solution of 3-(2-dithiopyridyl)propionic acid hydrazide in a water bath, the method further includes: taking the carboxyl magnetic bead product, shaking the carboxyl magnetic bead product, and then using a pipette to remove the fourth supernatant.

[0035] In one example, take 20 μL of the carboxylated magnetic bead product, approximately 20 mg, shake well, pipette the carboxylated magnetic beads, add them to a new EP tube, and discard the supernatant.

[0036] The carboxyl magnetic beads after the first water bath heating and dithiothreitol (DTT) are subjected to a second water bath heating to obtain carboxyl magnetic beads after the second water bath heating (i.e., mercapto magnetic beads).

[0037] In one example, the volume of DTT in the second water bath heating was 100 uL and the concentration was 10 mM.

[0038] In some embodiments, the heating temperature of the second water bath heating is 37°C, and the heating time is 0.5 h.

[0039] After the carboxyl magnetic beads were first washed following the second water bath heating, 3-(2-dithiopyridyl)propionic acid hydrazide (PDPH) was added and shaken to obtain chemically cleavable hydrazide magnetic beads.

[0040] In some embodiments, the first cleaning involves rinsing three times with ultrapure water.

[0041] In some embodiments, the volume of PDPH is 100 μL.

[0042] In some embodiments, the oscillation temperature is 37°C and the oscillation time is 1 hour.

[0043] After a second cleaning of the chemically cleavable hydrazine beads, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and small molecules (i.e. Figure 1 Small molecule ligands in the solution are subjected to a third water bath heating to obtain chemically cleavable small molecule magnetic beads.

[0044] In this embodiment, based on the covalent bonding of acylhydrazine chemistry, the acylhydrazine group in chemically cleavable acylhydrazine magnetic beads is used as the reactive group for covalent bonding with small molecules. This covalent bonding reaction has advantages such as simplicity, speed, low material cost and easy availability, strong scalability, and applicability to a variety of small molecules. Acylhydrazine is a chemically flexible and stable group that can be stored for a long time and reacts with various functional groups (such as carboxyl, carbonyl, phosphate groups, and sugars) under relatively simple reaction conditions or catalyzed by water-soluble carbodiimide as a coupling agent. Metabolites containing carbonyl, carboxyl, and phosphate groups, as well as small sugar molecules, account for a large proportion of the overall metabolome and play a key role in central carbon metabolism and energy metabolism.

[0045] In this embodiment, based on the chemically cleavable properties of disulfide bonds, a disulfide bond is designed on a chemically cleavable small molecule magnetic bead. This disulfide bond can be opened under the action of a reducing agent, thereby rapidly releasing the small molecule and its protein receptor, facilitating further identification. This process only releases the small molecule ligand and protein receptor, effectively avoiding interference from non-specifically bound proteins on the magnetic bead.

[0046] In this embodiment, chemically cleavable hydrazide magnetic beads can be fixed using a magnetic frame. In this embodiment, small molecules are covalently attached to the magnetic beads. By using magnetic beads for fixation, excess reagents in each reaction step can be easily removed, thus simplifying the entire synthesis process. This method enables the rapid synthesis of hundreds or even thousands of chemically cleavable small molecule magnetic beads.

[0047] In some embodiments, the second cleaning involves rinsing three times with ultrapure water.

[0048] In some embodiments, the volume of EDC added after the second wash is 50 μL, the volume of the small molecule is 50 μL, and the final concentration is 100 mM.

[0049] In some embodiments, the small molecule includes a small molecule containing a carboxyl group, a carbonyl group, or a phosphate group.

[0050] In some embodiments, the small molecule includes myristic acid. Myristic acid is a small molecule containing a carboxyl group.

[0051] In some embodiments, the heating temperature of the third water bath heating is 37°C, and the heating time is 1 hour.

[0052] After a third wash of the chemically cleavable small molecule magnetic beads, a protein mixture is added and incubated. Figure 2 As shown, Figure 2 Small molecule magnetic beads are Figure 1 The chemically cleavable small molecule magnetic beads are incubated with a protein mixture after a third washing of the beads. Figure 2 The structure in which the small molecule ligand binds to the protein receptor is the structure after incubation.

[0053] In some embodiments, the protein mixture is a protein mixture dissolved in phosphate buffer.

[0054] In some embodiments, the incubation time is 2 hours.

[0055] In some embodiments, the third cleaning involves rinsing three times with ultrapure water.

[0056] After incubation, the second supernatant was removed to obtain incubated magnetic beads; after a fourth wash of the incubated magnetic beads, a reducing agent and an auxiliary elution reagent were added, followed by a fourth water bath heating to elute interacting proteins, and the third supernatant was collected. Figure 2 In the process, a reducing agent (not labeled as an elution aid in the figure) is added to the incubated structure to elute the interacting proteins.

[0057] In some embodiments, the reducing agent is dithiothreitol; the auxiliary elution reagent is urea.

[0058] In this embodiment, dithiothreitol and urea are used together as eluents for elution. If the eluent only contains dithiothreitol, elution will be incomplete due to steric hindrance. However, by adding urea, the elution efficiency can be improved by urea assisting in elution.

[0059] In some embodiments, the concentration of dithiothreitol as the reducing agent is 10-100 mM, and the concentration of urea as the auxiliary elution reagent is 8 M.

[0060] In some embodiments, the heating temperature of the fourth water bath heating is 25-37°C, and the heating time is 0.5-2 hours.

[0061] In some embodiments, the fourth water bath heating temperature is 37°C and the heating time is 1 hour.

[0062] The third supernatant was used as the eluent for mass spectrometry identification. Figure 2 Mass spectrometry is used to identify large-scale small molecule interacting proteins.

[0063] In some embodiments, the step of performing mass spectrometry identification includes: adding chloroacetamide (CAA) to the eluent to react and obtain a reactant to block disulfide bonds; adding NH4HCO3 and trypsin to the reactant for enzymatic digestion to obtain an enzyme-digested sample; and performing mass spectrometry identification on the enzyme-digested sample after desalting.

[0064] For example, the concentration of CAA is 50 mM. The reaction of adding chloroacetamide (CAA) to the eluent is carried out at a temperature of 37°C for 1 hour.

[0065] In some embodiments, NH4HCO3 and trypsin are added to the reactants, and the mixture is enzymatically hydrolyzed overnight at 37°C to obtain an enzymatically digested sample.

[0066] In this embodiment, firstly, chemically cleavable hydrazide magnetic beads, i.e., hydrazide magnetic bead materials containing disulfide bonds, are designed and synthesized. Next, this magnetic bead material is covalently linked to a corresponding small molecule to prepare chemically cleavable small molecule magnetic beads, achieving large-scale synthesis of small molecule magnetic beads. Finally, these small molecule magnetic beads can be incubated with a non-denatured protein mixture and eluted under the action of a reducing agent. Subsequently, large-scale mass spectrometry identification is used to determine the receptor protein interacting with the small molecule.

[0067] This invention provides a method for identifying interactions between small molecules and proteins. The method involves synthesizing chemically cleavable hydrazide magnetic beads, specifically hydrazide magnetic beads containing disulfide bonds. The magnetic beads are then covalently linked to small molecules using amide chemistry to prepare chemically cleavable small molecule magnetic beads. The small molecule magnetic beads are incubated with a non-denatured protein mixture and eluted using a reducing agent and an elution aid. Mass spectrometry is then used to identify the receptor protein interacting with the small molecule. The chemically cleavable nature of the disulfide bonds in this method allows for rapid release of only the small molecule and its protein receptor, facilitating subsequent identification and avoiding non-specific binding. Furthermore, the chemically cleavable hydrazide magnetic beads facilitate immobilization, simplifying the removal of excess reagents and enabling the rapid synthesis of thousands of small molecule magnetic beads.

[0068] The methods in the embodiments of this application will be described in detail below through specific examples and comparative examples.

[0069] Example 1

[0070] Take a 20 μL volume (approximately 20 mg) of carboxyl magnetic beads, shake well, and pipette it into a new EP tube, discarding the supernatant. Add 50 μL of EDC aqueous solution and 50 μL of LPDPH aqueous solution, and heat in a water bath at 37°C for 1 h. Discard the supernatant, add 100 μL of 10 mM MDT, and heat in a water bath at 37°C for 0.5 h. Wash three times with ultrapure water. Add 100 μL of PDPH and shake at 37°C for 1 h to obtain chemically cleavable hydrazide magnetic beads. Wash three times with ultrapure water. Add 50 μL of EDC and 50 μL of a small molecule (final concentration maintained at 100 mM, myristic acid in this example), and heat in a water bath at 37°C for 1 h. Wash three times with ultrapure water. The protein mixture was dissolved in 500 μg phosphate buffer and incubated for 2 hours. The supernatant was removed, and the mixture was washed three times with ultrapure water. 20 μL of 100 mM DTT and 8 M urea were added, and the mixture was heated in a water bath at 37°C for 1 hour to elute the interacting proteins. The supernatant was collected and used as the eluent. The eluent was stained with Coomassie Brilliant Blue. Mass spectrometry was performed on the eluent, specifically by adding 50 mM CAA to the eluent and reacting at 37°C for 1 hour to obtain a reactant to block disulfide bonds. 200 μL of NH4HCO3 and 0.5 μL of trypsin (approximately 0.25 μg) were added to the reactant, and the mixture was digested overnight at 37°C to obtain the enzyme-digested sample. The enzyme-digested sample was desalted and then identified by mass spectrometry.

[0071] Example 2

[0072] Unlike Example 1, 50 μL of small molecules were not added.

[0073] Example 3

[0074] Unlike Example 1, 100 mM DTT was not added.

[0075] Example 4

[0076] Unlike Example 1, 100 mM DTT and 50 μL of small molecules were not added.

[0077] Example 5

[0078] Unlike Example 1, no 8M urea was added.

[0079] Example 6

[0080] Unlike Example 1, no 8M urea or 50ul of small molecules were added.

[0081] Example 7

[0082] Unlike Example 1, 50 μL of small molecules were not added. After eluting the interacting proteins by adding 20 μL of 100 mM DTT and 8 M urea and heating in a water bath at 37°C for 1 h, the supernatant was not collected and used as the eluent. Instead, 20 μL of 100 mM DTT and 8 M urea were directly added and heated in a water bath at 37°C for 1 h to elute the interacting proteins. Then, 50 mM CAA was added and reacted at 37°C for 1 h to obtain the reactant for blocking disulfide bonds. 200 μL of NH4HCO3 and 0.5 μL of trypsin (approximately 0.25 μg) were added to the reactant, and the mixture was incubated overnight at 37°C to obtain the digested sample. The digested sample was desalted and identified by mass spectrometry.

[0083] Figure 3 A schematic diagram of the Coomassie brilliant blue staining results of Examples 1-6 provided according to some embodiments is shown as an example. Figure 3 The content in the table, viewed from right to left, shows the elution effect of Example 1 (i.e., the elution effect with myristic acid listed under 100 mM dithiothreitol and 8 M urea), Example 2 (i.e., the elution effect with the control group listed under 100 mM dithiothreitol and 8 M urea), Example 3 (i.e., the elution effect with myristic acid listed under 8 M urea), Example 4 (i.e., the elution effect with the control group listed under 8 M urea), Example 5 (i.e., the elution effect with myristic acid listed under 100 mM dithiothreitol), and Example 6 (i.e., the elution effect with the control group listed under 100 mM dithiothreitol). It can be seen that Example 1 has the best elution effect, thus indicating that the elution effect is best when both dithiothreitol and urea are used as the eluent.

[0084] Table 1 shows partial identification results for Examples 2 and 7. Example 7 presents the results of direct enzymatic digestion on the magnetic beads, i.e., non-chemical cleavage elution. All proteins on the magnetic beads, including both specifically bound and non-specifically bound proteins, will be sent for mass spectrometry identification. The results show that Example 2, as the control group (i.e., no small molecules were attached to the magnetic beads), had zero non-specifically bound proteins. In contrast, Example 7, as the control group for enzymatic digestion on the magnetic beads (i.e., no small molecules were attached to the magnetic beads), showed a significant number of non-specifically bound proteins being sent to the mass spectrometer. Therefore, it is evident that the chemically cleavable small molecule magnetic beads in this application can effectively reduce non-specific binding.

[0085] Table 1

[0086]

[0087] It will be readily understood by those skilled in the art that the above-described advantageous methods can be freely combined and superimposed without conflict. The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. The above are merely preferred embodiments of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the protection scope of this application.

Claims

1. A method for identifying small molecule and protein interactions, characterized in that, The application relates to a method for preparing a chemically cleavable small molecule magnetic bead. The method comprises the following steps: a first water bath heating of carboxyl magnetic beads, 1-ethyl-(3-dimethylamine propyl) carbodiimide hydrochloride aqueous solution and 3-(2-dithio pyridyl) propionic acid hydrazine aqueous solution, and discarding a first supernatant to obtain the first water bath heated carboxyl magnetic beads; a second water bath heating of the first water bath heated carboxyl magnetic beads and dithiothreitol to obtain the second water bath heated carboxyl magnetic beads; after a first washing of the second water bath heated carboxyl magnetic beads, 3-(2-dithio pyridyl) propionic acid hydrazine is added for oscillation to obtain chemically cleavable hydrazine magnetic beads; after a second washing of the chemically cleavable hydrazine magnetic beads, 1-ethyl-(3-dimethylamine propyl) carbodiimide hydrochloride and small molecules are added, and a third water bath heating is performed to obtain chemically cleavable small molecule magnetic beads; after a third washing of the chemically cleavable small molecule magnetic beads, a protein mixture is added for incubation; after the incubation, a second supernatant is removed to obtain the incubated magnetic beads; after a fourth washing of the incubated magnetic beads, a reducing agent and an auxiliary elution reagent are added for a fourth water bath heating to elute the interacting proteins, and a third supernatant is collected; the third supernatant is used as an eluent to perform mass spectrometric identification; the small molecules comprise small molecules containing carboxyl, carbonyl or phosphate groups; 2. The method of claim 1, wherein, the heating temperature of the first water bath heating is 4-37 DEG C, and the heating time is 0.5-2h; the heating temperature of the second water bath heating is 37 DEG C, and the heating time is 0.5h; the heating temperature of the third water bath heating is 37 DEG C, and the heating time is 1h; the heating temperature of the fourth water bath heating is 25-37 DEG C, and the heating time is 0.5-2h.

3. The method of claim 1, wherein, the step of performing mass spectrometric identification comprises: adding chloroacetyl amide into the eluent for reaction to obtain a reaction product to block the disulfide bond; adding NH4HCO3 and trypsin into the reaction product for enzymolysis to obtain an enzyme digestion sample; and performing mass spectrometric identification on the desalted enzyme digestion sample.

4. The method of claim 3, wherein, the reducing agent is dithiothreitol; and the auxiliary elution reagent is urea.

5. The method of claim 1, wherein, The concentration of dithiothreitol as the reducing agent is 10-100mM, and the concentration of urea as the auxiliary elution reagent is 8M.

6. The method of claim 1, wherein, The protein mixture is a protein mixture dissolved in a phosphate buffer.

7. The method of claim 1, wherein, The small molecules comprise myristic acid.

8. The method of claim 1, wherein, The first washing, the second washing, the third washing and the fourth washing all adopt ultrapure water washing for three times. Before the step of water bath heating of the carboxyl magnetic beads, 1-ethyl-(3-dimethylamine propyl) carbodiimide hydrochloride aqueous solution and 3-(2-dithio pyridyl) propionic acid hydrazine aqueous solution, the method further comprises the following steps: taking a carboxyl magnetic bead product, shaking the carboxyl magnetic bead product, and using a pipette to suck the carboxyl magnetic beads to remove a fourth supernatant.

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