WO2018233328A1

WO2018233328A1 - Protein loading buffer and use thereof in preparation of protein chip

Info

Publication number: WO2018233328A1
Application number: PCT/CN2018/079494
Authority: WO
Inventors: Guoxin Wang; Tao LIAO; Minwen CHEN; Meijie TANG; Su Zhao
Original assignee: Wwhs Biotech, Inc; Nirmidas Biotech, Inc.
Priority date: 2017-06-22
Filing date: 2018-03-19
Publication date: 2018-12-27
Also published as: CN107167613B; CN107167613A

Abstract

A protein loading buffer and a use thereof in preparation of a protein chip, particularly a plasmonic gold chip, in which the protein loading buffer includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.

Description

PROTEIN LOADING BUFFER AND USE THEREOF IN PREPARATION OF PROTEIN CHIP

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims a priority to and benefits of Chinese Patent Application Serial No. 201710482189.6, filed with the State Intellectual Property Office of P.R. China on June 22, 2017, the entire content of which is incorporated herein by reference.

FIELD

The present disclosure relates to the field of biotechnology, in particular, to a protein loading buffer and use thereof in preparation of a protein chip, particularly a plasmonic gold chip.

BACKGROUND

Plasmonic materials are typically referred to noble metals (e.g. gold) and their hybrids, which enjoy unique surface plasmon resonance under light irradiation within specific wavelength ranges. With tailored structural parameters and surface chemistry, plasmonic materials engaged with near-infrared fluorescence enhanced (NIR-FE, 650-1700 nm) applications and achieved detection of biomarkers through micro-spotting of selected probes (eg. an antigen or antibody) on surface in the chip format.

At present, immunoassay methods for clinical application mainly include immunonephelometry, immunochromatography, enzyme linked immunosorbent assay (ELISA) , chemiluminescence immunoassay (CLIA) , and protein chip-immunofluorescence assay which has been developed in recent years. These protein chip assays all involve a critical technology to maintain stability of proteins on the chip, as a trace amount of bioactive substance (eg. an antigen or antibody) is liable to loss its activity after immobilized on a chip.

Therefore, it is of significance to maintain stability of proteins on the chip.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art at least to some extent. An object of the present disclosure is to provide a protein loading buffer and use thereof in preparation of a protein chip, particularly a plasmonic gold chip. The protein loading buffer is capable of maintaining bioactivity of a protein immobilized on a chip for a long term; improving immobilization between the protein and the chip; and shortening incubation period of the protein after loaded.

In a first aspect, the present disclosure provides in embodiments a protein loading buffer, including trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.

In some embodiments, the protein loading buffer of the present disclosure includes trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate. Compared to a glycerin or phosphate buffer widely used at present, the protein loading buffer is capable of not only allowing a protein to be immobilized on a chip substrate in a stronger way, but also shortening incubation period of the protein after loaded, as well as maintaining bioactivity of the protein immobilized for a longer term.

In some embodiments, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 20 g/L, the glycine is of a concentration of 5 g/L to 10 g/L, the isoleucine is of a concentration of 5 g/L to 10 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 1 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.2 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded, and maintaining bioactivity of the protein immobilized for a long term.

In some embodiments, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 18 g/L, the glycine is of a concentration of 5 g/L to 9 g/L, the isoleucine is of a concentration of 5 g/L to 9 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.9 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.15 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In some embodiments, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 15 g/L, the glycine is of a concentration of 5 g/L to 7 g/L, the isoleucine is of a concentration of 5 g/L to 7 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.7 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.12 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In some embodiments, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L, the glycine is of a concentration of 5 g/L, the isoleucine is of a concentration of 5 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In some embodiments, the protein loading buffer is of a pH value of 6.0 to 7.2.

In some embodiments, the protein loading buffer is of a pH value of 6.0.

In some embodiments, the pH value of the protein loading buffer is adjusted with at least one of a sodium hydroxide solution and a hydrochloric acid solution, thus adjusting the pH value of the protein loading buffer effectively.

In another aspect, the present disclosure provides in embodiments use of a protein loading buffer described in the first aspect in preparation of a protein chip.

In some embodiments, the protein chip includes a plasmonic gold chip.

In some embodiments, the plasmonic gold chip includes a glass substrate and a nanogold layer coated on a surface of the glass substrate. Therefore, a selected protein can be immobilized onto the plasmonic gold chip in a predetermined sequence through loading technology, so as to achieve detection of biomarkers in a simple way.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

DESCRIPTION OF DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the drawings, in which:

Figure 1 are fluorescent scanning graphs showing comparison among CEA antibodies loaded with different protein loading buffers after storage for 1 day, 3 days, 5 days and 7 days at 37℃, respectively, according to examples 1-5 and comparative examples 1-2 of the present disclosure.

Figure 2 are histograms of fluorescence intensities of CEA antibodies loaded with different protein loading buffers after storage for 1 day, 3 days, 5 days and 7 days at 37℃, respectively, according to example 1 and comparative examples 1-2 of the present disclosure.

Figure 3 are fluorescent scanning graphs showing comparison among CEA antibodies loaded with different protein loading buffers after incubation for 0.5 hours, 6 hours and 12 hours at 30℃, respectively, according to examples 1-5 and comparative examples 1-2 of the present disclosure.

Figure 4 are histograms of fluorescence intensities of CEA antibodies loaded with different protein loading buffers after incubation for 0.5 hours, 6 hours and 12 hours at 30℃, respectively, according to example 1 and comparative examples 1-2 of the present disclosure.

DETAILED DESCRIPTION

Embodiments of the present disclosure are described in detail below, and examples of the embodiments are shown in the accompanying drawings, in which like or similar reference numerals refer to like or similar elements or elements having the same or similar functions throughout the description. The embodiments described below with reference to the accompanying drawings are exemplary, which are intended to be illustrative of the disclosure and are not to be construed as a limit of the disclosure.

The present disclosure is accomplished by the present inventors based on the following discoveries.

At present, a protein loading buffer wildly used in preparation of a protein chip, particularly a plasmonic gold chip includes a glycerin or phosphate buffer, which is poor in maintaining bioactivity of a protein on the protein chip effectively for a long term. The protein chip was usually frozen at a temperature of -20℃ or below for a long-term storage and avoided repeated freezing and thawing cycles in the prior art to maintain bioactivity of a protein on the protein chip, while placed in a refrigerator at 2℃ to 8℃ for a week or placed at 37℃ for a day, with a greater than 50%loss in bioactivity of the protein. In addition, in order to be immobilized on a protein chip, a protein should be incubated for 12 to 24 hours after loaded onto the protein chip by common methods. It is surprisingly found by the present inventors that a protein loading buffer formulated with trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate can solve at least one of the problems existed in the prior art, such as poor immobilization between a protein and a chip substrate, significant loss in bioactivity of the protein immobilized and so on.

Thus, in a first aspect, the present disclosure in embodiments provides a protein loading buffer, including trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate. Compared to a glycerin or phosphate buffer widely used at present, the protein loading buffer is capable of not only allowing a protein to be immobilized on a chip substrate in a stronger way, but also shortening incubation period of the protein after loaded, as well as maintaining bioactivity of the protein immobilized for a longer term.

In specific embodiments of the present disclosure, the protein loading buffer is an aqueous solution including trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate. When used in preparation of a protein chip, for example, a plasmonic gold chip, the protein loading buffer of the present disclosure is capable of moisturizing a protein on the plasmonic gold chip, thus preventing the protein from inactivating owing to over-drying; allowing a protein to be immobilized on a chip substrate in a stronger way; shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a longer term, as compared to a glycerin or phosphate buffer widely used in the prior art.

In specific embodiments of the present disclosure, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 20 g/L, the glycine is of a concentration of 5 g/L to 10 g/L, the isoleucine is of a concentration of 5 g/L to 10 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 1 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.2 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In specific embodiments of the present disclosure, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 18 g/L, the glycine is of a concentration of 5 g/L to 9 g/L, the isoleucine is of a concentration of 5 g/L to 9 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.9 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.15 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In specific embodiments of the present disclosure, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L to 15 g/L, the glycine is of a concentration of 5 g/L to 7 g/L, the isoleucine is of a concentration of 5 g/L to 7 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.7 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.12 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In a specific embodiment of the present disclosure, in the protein loading buffer of the present disclosure, the trehalose is of a concentration of 10 g/L, the glycine is of a concentration of 5 g/L, the isoleucine is of a concentration of 5 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L, thus further improving immobilization between the protein and the chip substrate, shortening incubation period of the protein after loaded; and maintaining bioactivity of the protein immobilized for a long term.

In specific embodiments of the present disclosure, the protein loading buffer is of a pH value of 6.0 to 7.2, thereby further maintaining bioactivity of the protein immobilized on the chip substrate for a long term.

In specific embodiments of the present disclosure, the protein loading buffer is of a pH value of 6.0, thereby further maintaining bioactivity of the protein immobilized on the chip substrate for a long term.

In specific embodiments of the present disclosure, the pH value of the protein loading buffer is adjusted with at least one of a sodium hydroxide solution and a hydrochloric acid solution. Thus, by controlling concentration and addition amounts of the sodium hydroxide solution and/or the hydrochloric acid solution, the protein loading buffer of the present disclosure is provide with an optimal pH value.

In specific embodiments of the present disclosure, the protein chip includes a plasmonic gold chip.

In specific embodiments of the present disclosure, the plasmonic gold chip includes a glass substrate and a nanogold layer coated on a surface of the glass substrate. Therefore, a selected protein can be immobilized onto the plasmonic gold chip in a predetermined sequence through loading technology, so as to achieve detection of biomarkers in a simple way.

In specific embodiments of the present disclosure, the use of the protein loading buffer described above improves immobilization between the protein and the substrate of the plasmonic gold chip effectively, thereby shortening incubation period of the protein loaded onto the plasmonic gold chip, particularly, 0.5 hours; and maintains bioactivity of the protein immobilized on the plasmonic gold chip effectively, particularly, storage at 37℃ for 7 days, with 80%or greater bioactivity of the protein retained relative to initial bioactivity.

In the following, the technical solution of the present disclosure will be described in detail in combination with examples. It should be appreciated to those skilled in the art that, the examples described below are explanatory, illustrative, and used to generally understand the present disclosure, and shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions. Examples which do not indicate specific techniques or conditions are carried out either in accordance with the techniques or conditions described in the literatures in the related art or in accordance with the product specifications. The reagents or instruments whose manufacturers are not indicated are all conventional products, which are commercially available.

EXAMPLES

Inventive Example 1

A protein loading buffer was formulated with the following components in respective final concentrations: 10 g/L trehalose, 5 g/L glycine, 5 g/L isoleucine, 0.2 mol/L ammonium sulfate and 0.05 mol/L sodium citrate, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 6.0, and stored at 2℃ to 8℃ for future use.

Inventive Example 2

A protein loading buffer was formulated with the following components in respective final concentrations: 15 g/L trehalose, 6 g/L glycine, 7 g/L isoleucine, 0.4 mol/L ammonium sulfate and 0.12 mol/L sodium citrate, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 6.5, and stored at 2℃ to 8℃ for future use.

Inventive Example 3

A protein loading buffer was formulated with the following components in respective final concentrations: 12 g/L trehalose, 7 g/L glycine, 6 g/L isoleucine, 0.7 mol/L ammonium sulfate and 0.09 mol/L sodium citrate, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 7.2, and stored at 2℃ to 8℃ for future use.

Inventive Example 4

A protein loading buffer was formulated with the following components in respective final concentrations: 18 g/L trehalose, 8 g/L glycine, 8 g/L isoleucine, 0.9 mol/L ammonium sulfate and 0.15 mol/L sodium citrate, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 6.0, and stored at 2℃ to 8℃ for future use.

Inventive Example 5

A protein loading buffer was formulated with the following components in respective final concentrations: 19 g/L trehalose, 10 g/L glycine, 10 g/L isoleucine, 1 mol/L ammonium sulfate and 0.18 mol/L sodium citrate, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 6.0, and stored at 2℃ to 8℃ for future use.

Comparative Example 1

A protein loading buffer was formulated with the following components in respective final concentrations: 5% (v/v) glycerin and 10 mM PBS, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 6.0, and stored at 2℃ to 8℃ for future use.

Comparative Example 2

A protein loading buffer was formulated with PBS in a final concentration of 10 mM, which was adjusted with 1 mol/L sodium hydroxide solution and/or 1 mol/L hydrochloric acid solution to be of a pH value of 7.2, and stored at 2℃ to 8℃ for future use.

Testing Example 1

Protective effect of the protein loading buffers of inventive examples 1 to 5 and comparative examples 1-2 on bioactivity of Carcino Embryonic Antigen (CEA) capturing antibody immobilized on a plasmonic gold chip

1) Loading CEA capturing antibody onto a plasmonic gold chip

The CEA capturing antibody (Shanghai Linc-Bio Science Co. LTD) was diluted to 0.2 mg/mL with each of the protein loading buffers prepared in inventive examples 1 to 5 and comparative examples 1 and 2, then printed onto wells from the first row to the seventh row of a plasmonic gold chip by GeSim Nano-Plotter ^TM 2.1, with 3 nL and 5 replicates for one point, which became a round spot in a diameter of about 400 microns.

2) Preparing a plasmonic gold chip to be tested

Such seven plasmonic gold chips loaded with CEA capturing antibody were incubated at room temperature for 0.5 hours, and then stored in vacuum at 37℃. On 1 day, 3 days, 5 days and 7 days after storage, one plasmonic gold chip was taken out for detection at each time.

Specifically, a plasmonic gold chip to be tested was prepared as follows.

The plasmonic gold chip loaded with CEA capturing antibody after storage, was blocked with 1%BSA in PBS under shaken for 1 hour to reduce nonspecific binding, then washed by PBST with 150 μL/well (0.05%Tween 20) , and subsequently added with 100 μL CEA antigen (10 ng/mL) (Shanghai Linc-Bio Science Co. LTD, L2C010) to each well and shaken for 2 hours. After washed with PBST for three times, the plasmonic gold chip was added with CEA antibody labeled with IRDye800 (4 nmol/L) (LI-COR Corporate, P/N 929-7002) to stain for 0.5 hours in the dark under stirring, followed by successively washed with PBST three times and with pure water once, and then centrifugal dried, thus obtaining the plasmonic gold chip to be tested.

3) Fluorescence quantification and analysis of detection results

The plasmonic gold chip to be tested was scanned with MidaScan scanner at 800 nm channel under a laser intensity of 7.0 and a resolution of 20 μm. After scanning, 16-bit gray-scale images were obtained and analyzed with MidaScan Software V1.0.0 or a higher version. The intensity of each point was measured by gate array analysis mode and the lattice morphology was automatically recognized by the program. The intensity of each point was obtained by dividing the total signal strength of a selected region by area of the selected region. The average fluorescence intensity of the five parallel points on the image was defined as the measured intensity. There was a positive correlation between the activity of CEA capture antibody and the fluorescence intensity on the obtained image.

The detection results are shown in Table 1 and Figures 1-2. Table 1 shows fluorescence intensities of CEA capturing antibodies loaded with different protein loading buffers after storage for 1 day, 3 days, 5 days and 7 days at 37℃. Figures 1-2 respectively show comparison among fluorescent scanning graphs and fluorescence intensity histograms of CEA capturing antibodies loaded with different protein loading buffers after storage for 1 day, 3 days, 5 days and 7 days at 37℃, respectively.

Table 1 fluorescence intensities of CEA capturing antibodies loaded with different protein loading buffers after storage for 1 day, 3 days, 5 days and 7 days at 37℃

Testing Example 2

Effect of the protein loading buffers of inventive examples 1-5 and comparative examples 1-2 on immobilization between the CEA capturing antibody and the substrate of the plasmonic gold chip

1) Loading CEA capturing antibodies onto a plasmonic gold chip

The CEA capturing antibody was loaded onto the plasmonic gold chip as similar to those in the testing example 1.

2) Preparing plasmonic gold chips to be tested

Such seven plasmonic gold chips loaded with CEA capturing antibody were incubated at 30℃ for 0.5 hours, 6 hours and 12 hours respectively, after which each plasmonic gold chip was taken out for detection.

Specifically, plasmonic gold chips to be tested were prepared as follows.

The plasmonic gold chips loaded with CEA capturing antibody after incubation were blocked with 1%BSA in PBS under shaken for 1 hour to reduce nonspecific binding, then washed by PBST with 150 μL/well (0.05%Tween 20) , next added with 100 μL CEA antigen (10 ng/mL) (Shanghai Linc-Bio Science Co. LTD, L2C010) to each well and shaken for 2 hours. After washed with PBST for three times, the plasmonic gold chips were added with CEA antibody labeled with IRDye800 (4 n mol/L) (LI-COR Corporate, P/N 929-7002) to stain for 0.5 hours in the dark under stirring, followed by successively washed with PBST three times and with pure water once, and then centrifugal dried, thus obtaining the plasmonic gold chips to be tested.

3) Fluorescence quantification and analysis of detection results

The fluorescence intensities of the plasmonic gold chips to be tested were quantified and the detection results were analyzed as similar to those in the testing example 1.

The detection results are shown in Table 2 and Figures 3-4. Table 2 shows fluorescence intensities of CEA capturing antibodies loaded with different protein loading buffers after incubation for 0.5 hours, 6 hours and 12 hours at 30℃ respectively. Figures 3-4 respectively show comparison among fluorescent scanning graphs and fluorescence intensity histograms of CEA capturing antibodies loaded with different protein loading buffers after incubation for 0.5 hours, 6 hours and 12 hours at 30℃ respectively.

Table 2 fluorescence intensities of CEA capturing antibodies loaded with different protein loading buffers after incubation for 0.5 hours, 6 hours and 12 hours at 30℃

Conclusion:

1) It can be concluded from Table 1 and Figures 1-2 that the protein loading buffers in inventive examples 1-5, after incubation of the plasmonic gold chip loaded with CEA capturing antibody for 7 days at 37℃, maintains the bioactivity of the CEA capturing antibody on the chip effectively, with 80%or greater bioactivity of the protein retained relative to initial bioactivity.

2) It can be concluded from Table 2 and Figures 3-4 that the protein loading buffers in inventive examples 1-5, after incubation of the plasmonic gold chip loaded with CEA capturing antibody for 0.5 hours, improves immobilization between the CEA capturing antibody and the substrate of the plasmonic gold chip effectively, and shortens incubation period for immobilization.

Reference throughout this specification to “an embodiment” , “some embodiments” , “one embodiment” , “another example” , “an example” , “a specific example” or “some examples” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments” , “in one embodiment” , “in an embodiment” , “in another example” , “in an example” , “in a specific example” or “in some examples” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A protein loading buffer, comprising trehalose, glycine, isoleucine, ammonium sulfate and sodium citrate.
The protein loading buffer according to claim 1, wherein the trehalose is of a concentration of 10 g/L to 20 g/L, the glycine is of a concentration of 5 g/L to 10 g/L, the isoleucine is of a concentration of 5 g/L to 10 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 1 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.2 mol/L.
The protein loading buffer according to claim 1 or 2, wherein the trehalose is of a concentration of 10 g/L to 18 g/L, the glycine is of a concentration of 5 g/L to 9 g/L, the isoleucine is of a concentration of 5 g/L to 9 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.9 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.15 mol/L.
The protein loading buffer according to any one of claims 1 to 3, wherein the trehalose is of a concentration of 10 g/L to 15 g/L, the glycine is of a concentration of 5 g/L to 7 g/L, the isoleucine is of a concentration of 5 g/L to 7 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L to 0.7 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L to 0.12 mol/L.
The protein loading buffer according to any one of claims 1 to 4, wherein the trehalose is of a concentration of 10 g/L, the glycine is of a concentration of 5 g/L, the isoleucine is of a concentration of 5 g/L, the ammonium sulfate is of a concentration of 0.2 mol/L, and the sodium citrate is of a concentration of 0.05 mol/L.
The protein loading buffer according to any one of claims 1 to 5, wherein the protein loading buffer is of a pH value of 6.0 to 7.2.
The protein loading buffer according to any one of claims 1 to 6, wherein the protein loading buffer is of a pH value of 6.0.
The protein loading buffer according to claim 6 or 7, wherein the pH value is adjusted with at least one of a sodium hydroxide solution and a hydrochloric acid solution.
Use of a protein loading buffer according to any one of claims 1 to 8 in preparation of a protein chip.
The use according to claim 9, wherein the protein chip comprises a plasmonic gold chip.
The use according to claim 9 or 10, wherein the plasmonic gold chip comprises a glass substrate and a nanogold layer coated on a surface of the glass substrate.