Concentrated SDS-page loading buffer: compositions, methods of preparation, and applications

Abstract

Current loading buffers for SDS-PAGE often fail to effectively solubilize and stabilize hydrophobic proteins, such as membrane proteins. Due to their hydrophobic domains, these proteins tend to aggregate or precipitate in standard buffers, resulting in poor resolution, incomplete denaturation, and inaccurate quantification during electrophoresis. Most existing formulations are optimized for hydrophilic proteins, which leaves a performance gap when analyzing hydrophobic proteins. Thus, to overcome these challenges a novel loading buffer is described herein that enhances the solubility and stability of hydrophobic proteins, significantly improving the accuracy and reliability of their analysis.

Description

September 25, 2025CONCENTRATED SDS-PAGE LOADING BUFFER: COMPOSITIONS, METHODS OF PREPARATION, AND APPLICATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 700,242 filed September 27, 2024, the specification of which is incorporated herein in its entirety by reference.FIELD OF THE INVENTION

[0002] The present invention features a more concentrated solution of an SDS-PAGE sample loading buffer called 4X LOAD. The solution has higher concentrations of both a denaturing agent (e.g., SDS) and a reducing agent (e.g., beta-mercaptoethanol (BME)) compared to standard Laemmli and XT buffers. In some embodiments, the higher concentration of these components helps when it comes to running hydrophobic proteins (e.g., membrane proteins) on protein gels. The buffers described herein work just as well for SDS-PAGE and Western Blotting as both the standard loading buffers.BACKGROUND OF THE INVENTION

[0003] Cunent loading buffers used in protein analysis, particularly for SDS-PAGE, often fall short in effectively solubilizing and stabilizing hydrophobic proteins, such as membrane proteins. These proteins, characterized by their strong hydrophobic domains, tend to aggregate or precipitate in traditional buffers, leading to poor resolution, incomplete denaturation, or inaccurate quantification in electrophoretic techniques. Existing formulations are typically optimized for hydrophilic proteins, leaving a significant gap in performance for challenging hydrophobic proteins. Thus, the present invention addresses these limitations by generating a novel loading buffer composition specifically designed to enhance the solubility and stability of hydrophobic proteins, thereby improving the accuracy and consistency of their analysis.BRIEF SUMMARY OF THE INVENTION

[0004] It is an objective of the present invention to provide compositions and methods that allow for the denaturing of hydrophobic proteins, e.g., membrane proteins, as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0005] The present invention features a higher concentration loading buffer composition for useSeptember 25, 2025 with membrane proteins, since membrane proteins tend to be resistant to denaturation. The loading buffer composition described herein has higher concentrations of both the denaturing detergent (sodium dodecyl sulfate [SDS]) and the reducing agent (beta-mercaptoethanol [BME]) compared to the compositions of the following two widely referenced solutions: (i) Laemmli buffer, from 1970 Nature PMID 5432063 and (ii) Schagger Reducing sample Buffer A, from 2006 Nature Protocols PMID 17406207.

[0006] In some embodiments, the present invention features a loading buffer composition, e.g., an SDS loading buffer composition. In some embodiments, the composition comprises 10-15% wt / vol of a denaturing agent and 5-25% vol / vol of a reducing agent. In other embodiments, the composition comprises 12% wt / vol of a denaturing agent and 5-25% vol / vol of a reducing agent. In some embodiments, the composition comprises 10-15% wt / vol of a denaturing agent and 20% vol / vol of a reducing agent. In further embodiments, the composition comprises 12% wt / vol of a denaturing agent and 20% vol / vol of a reducing agent. In some embodiments, the denaturing agent is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate. In some embodiments, the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT). In some embodiments, the composition may further comprise one or a combination of water, Tris-HCl, glycerol, or bromophenol blue.

[0007] In other embodiments, the present invention features a method of denaturing a hydrophobic protein. In some embodiments, the method comprises obtaining or having obtained a protein sample, diluting the protein sample in the loading buffer composition as described herein, and heating the diluted protein sample for a period of time. In some embodiments, the hydrophobic protein is a membrane protein. In some embodiments, the protein sample is diluted in a 1 to 3 dilution; wherein 3 parts of the protein sample is diluted into one part of the loading buffer composition. In other embodiments, the protein sample is diluted in a 1 to 2 dilution; wherein 2 parts of the protein sample is diluted into one part of the loading buffer composition. In further embodiments, the protein sample is diluted in a 1 to 1 dilution; wherein 1 part of the protein sample is diluted into one part of the loading buffer composition.

[0008] In further embodiments, the present invention may feature a method of generating a loading buffer composition as described herein. In some embodiments, the method comprises a) adding water and Tris-HCl pH 6.8 to a tube, b) adding beta-mercaptoethanol to the tube and invert the tube at least once, c) adding glycerol to the tube and invert the tube at least once, d) adding bromophenol blue to the tube and invert the tube at least once, and e) adding SDS to the tube andSeptember 25, 2025 gently mix the tube to avoid creating bubbles. In some embodiments, the method may further comprise filtering the loading buffer composition. In some embodiments, the method further comprises discarding the first several drops upon filtering the loading buffer composition. The method may further comprise storing the loading buffer composition at -80°C, until use. In some embodiments, the loading buffer composition is viable at -20°C for up to 6 months

[0009] One of the unique and inventive technical features of the present invention is the use of a highly concentrated loading buffer. Without wishing to limit the invention to any theory or mechanism, it is believed that said technical feature advantageously provides for the ability to fully denature hydrophobic proteins, e.g., membrane proteins, better than commercially available loading buffers. None of the presently known prior references or works have the unique inventive technical feature of the present invention.

[0010] Moreover, the prior references teach away from the present invention. For example, the two widely referenced solutions have lower concentrations of either SDS or BME, and current commercial loading buffers are typically half as concentrated in both SDS and BME as the composition disclosed herein. Additionally, a person having ordinary skill in the art tends to rely on commercially available loading buffers, which are less concentrated and, thus, less effective for certain proteins. When encountering poor results, rather than addressing the inadequacy of the buffer, many will question the properties of the protein itself. However, these commercially available buffers are particularly ineffective for hydrophobic proteins, such as membrane proteins, or lipidated proteins, further underscoring the need for the present invention.

[0011] Furthermore, the inventive technical feature of the present invention contributed to a surprising result. Specifically, through the novel methods employed to formulate the loading buffer composition described herein, the Inventors were able to achieve a remarkably high concentration of both the denaturing agent and reducing agent, while maintaining their solubility even at room temperature. This outcome was particularly surprising, as such high concentrations would typically result in precipitation or instability under these conditions, highlighting the unique efficacy of the present invention. Without wishing to limit the present invention to any particular theory or mechanism, adding either or both the reducing agent (e.g., [3-mercaptoethanol) or glycerol prior to the denaturing agent (e.g., SDS), may help to achieve the high concentrations.

[0012] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one ofSeptember 25, 2025 ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0014] FIG. 1A and IB shows that the loading buffer composition described herein provides same results on SDS-PAGE as Bio-Rad® Laemmli & Bio-Rad® XT either stained using silver staining (FIG. 1A) or a Western (FIG. IB). Sample: Elution sample from immobilized metal affinity chromatography (IMAC; nickel-NTA) that was loaded with DDM-solubilized outer membrane of E. coli expressing BBA57-Hisl2 (55 kDa), a lipidated outer membrane protein. Samples were incubated at 95°C for 10 minutes in the loading buffer, which was at IX, 1.5X, 2X, or 3X concentration.

[0015] FIG. 2 shows a comparison of the denaturing capacity of different loading buffers at their IX concentration using the hydrophobic membrane protein FopA-His8 as a model substrate. The 4X LOAD buffer as described herein denatures FopA-His8 at a lower temperature than both the original 2X Laemmli buffer (Laemmli, Nature, 1970) and the commercial Bio-Rad® 4X Laemmli buffer (LDS-based), but not at a lower temperature than the commercial Bio-Rad 4X XT buffer (LDS-based, higher % LDS, proprietary composition). This figure also demonstrates a novel application of the heat modifiability characteristic of the FopA-His8 hydrophobic membrane protein to evaluate and compare the denaturing capacity of different loading buffer formulations. Heat modifiability, also referred to as mobility-shift, is a known characteristic of some beta-barrel membrane proteins, in which the protein remains incompletely denatured in a loading buffer when the protein is incubated in that loading buffer at one temperature, while upon increasing that incubation temperature, the protein becomes completely denatured, as indicated on SDS-PAGE by a shift of the protein to a higher molecular weight band. The application of the heat modifiability characteristic to evaluate and compare the denaturing capacity of different loading buffer formulations is novel.DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearlySeptember 25, 2025 indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessary solely". Furthermore, variation of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0017] Suitable methods and materials for the practice and / or testing of embodiments of the disclosure are described below. Such methods and materials are illustrative only and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which the disclosure pertains are described in various general and more specific references, including, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), "Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.), the disclosures of which are incorporated in their entirety herein by reference.

[0018] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.September 25, 2025

[0019] Although methods and materials similar or equivalent to those described herein can be used to practice or test the disclosed technology, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0020] Referring now to FIGs. 1A-2, the present invention features compositions and methods that allow for the denaturing of hydrophobic proteins, e.g., membrane proteins.

[0021] In some embodiments, the present invention features a loading buffer composition effective for denaturing hydrophobic proteins, e.g., an SDS loading buffer composition. In some embodiments, the composition comprises 10-15% wt / vol of a denaturing agent and 5-25% vol / vol of a reducing agent. In other embodiments, the composition comprises 12% wt / vol of a denaturing agent and 5-25% vol / vol of a reducing agent. In some embodiments, the composition comprises 10-15% wt / vol of a denaturing agent and 20% vol / vol of a reducing agent. In further embodiments, the composition comprises 12% wt / vol of a denaturing agent and 20% vol / vol of a reducing agent. In some embodiments, the denaturing agent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), or another ionic detergent suitable for protein denaturation. In some embodiments, the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT). In some embodiments, the composition may further comprise one or a combination of water, a buffer (Tris HC1 pH 6.8), glycerol, or a tracking dye (e.g., bromophenol blue or xylene cyanol, e.g., xylene cyanol FF). In other embodiments, the composition may further comprise one or a combination of water, Tris-HCl, glycerol, or bromophenol blue.

[0022] In other embodiments, the present invention features a method of denaturing a hydrophobic protein. In some embodiments, the method comprises obtaining or having obtained a protein sample, diluting the protein sample in the loading buffer composition as described herein, and heating the diluted protein sample for a period of time. In some embodiments, the hydrophobic protein is a membrane protein. In some embodiments, the protein sample is diluted in a 1 to 3 dilution; wherein 3 parts of the protein sample is diluted into one part of the loading buffer composition. In other embodiments, the protein sample is diluted in a 1 to 2 dilution; wherein 2 parts of the protein sample is diluted into one part of the loading buffer composition. In further embodiments, the protein sample is diluted in a 1 to 1 dilution; wherein 1 part of the protein sample is diluted into one part of the loading buffer composition.

[0023] In some embodiments, the diluted protein sample is heated to about 90 to 100°C. In some embodiments, the diluted protein sample is heated to about 92 to 98°C. In some embodiments, the diluted protein sample is heated to about 94 to 96°C. In some embodiments, the diluted proteinSeptember 25, 2025 sample is heated to about 95°C. In certain embodiments, the diluted protein sample is heated to a temperature of less than 90°C. For example, in some embodiments, the diluted protein sample is heated to a temperature of about 90°C, about 85°C, or about 80°C.

[0024] In some embodiments, the diluted protein sample is heated for about 5 to 20 minutes. In some embodiments, the diluted protein sample is heated for about 5 to 15 minutes. In some embodiments, the diluted protein sample is heated for about 10 minutes. In some embodiments, the diluted protein sample is heated for about 2 to 10 minutes. In some embodiments, the diluted protein sample is heated for about 4 to 8 minutes. In some embodiments, the diluted protein sample is heated for about 4 to 6 minutes. In some embodiments, the diluted protein sample is heated for about 5 minutes.

[0025] In certain embodiments, the present invention features a method of denaturing a hydrophobic protein (e.g., a membrane protein) at a temperature less than 90°C. In some embodiments, the method comprises obtaining or having obtained a protein sample, diluting the protein sample in the loading buffer composition as described herein, and heating the diluted protein sample for a period of time.

[0026] In further embodiments, the present invention provides a method for preparing a loading buffer composition as described herein. The method may comprise: (a) adding water and a buffering agent to a container, (b) adding a reducing agent, (c) adding a density agent such as glycerol, (d) adding an indicator dye, and (e) adding a denaturing agent such as sodium dodecyl sulfate (SDS). The components can be introduced sequentially in the order listed, simultaneously, or in any other suitable order, with mixing or inversion of the container after each addition as desired. In certain embodiments, the denaturing agent is preferably added after the glycerol and / or reducing agent. Without wishing to limit the present invention to any particular theory or mechanism, it is believed that adding SDS at this stage facilitates dissolution at the high concentrations used, potentially because the presence of glycerol and / or the reducing agent promotes solubilization.

[0027] In other embodiments, the method comprises a) adding water and Tris-HCl pH 6.8 to a tube, b) adding beta-mercaptoethanol to the tube and invert the tube at least once, c) adding glycerol to the tube and invert the tube at least once, d) adding bromophenol blue to the tube and invert the tube at least once, and e) adding SDS to the tube and gently mix the tube to avoid creating bubbles.

[0028] In some embodiments, the method may further comprise filtering the loading bufferSeptember 25, 2025 composition. In some embodiments, the method further comprises discarding the first several drops upon filtering the loading buffer composition. The method may further comprise storing the loading buffer composition at -80°C, until use. In some embodiments, the loading buffer composition is viable at -20°C for up to 6 months.

[0029] In some embodiments, the present invention features a method of detecting heat modifiability of a membrane protein. The method may comprise incubating a protein sample comprising the membrane protein in a loading buffer composition as described herein at a first incubation temperature, wherein at the first temperature the membrane protein remains incompletely denatured, incubating the protein sample in the loading buffer as described herein at a second temperature, wherein at the second temperature the membrane protein is completely denatured; and detecting a mobility shift of the protein on an SDS-PAGE gel, wherein the mobility shift is evidenced by migration of the protein to a higher molecular weight band at the second temperature as compared to the first temperature, thereby identifying the protein as heat modifiable.

[0030] Without wishing to limit the present invention to any theory or mechanism, the present invention provides systems, e.g., kits, for denaturing hydrophobic proteins that are designed to provide flexibility in use.

[0031] In some embodiments, the system comprises a pre-mixed solution that includes a denaturing agent, water, a buffer (e.g., Tris-HCl), glycerol, and a tracking dye, together with written instructions for preparation and / or use. In some embodiments, the pre-mixed solution is formulated without a reducing agent (e.g., 0-mercaptoethanol (BME)) to improve shelf stability, as reducing agents are prone to oxidation over time. In another embodiment, the system comprises a pre-mixed solution that includes the denaturing agent, water, a buffering component (e.g., Tris- HCl), glycerol, a tracking dye, and a reducing agent (e.g., BME). This embodiment is particularly suitable for laboratories with high usage, where frequent replenishment mitigates the potential degradation of the reducing agent. The inclusion of the reducing agent in the pre-mixed solution allows for immediate use without additional preparation steps.

[0032] In yet another embodiment, the system comprises separate containers each holding one or more of the following: a denaturing agent, a buffering agent, glycerol, a reducing agent, and a tracking dye; and instructions for preparing a loading buffer by combining the components in specified proportions.September 25, 2025

[0033] In certain variations of any of the above embodiments, the system may further include optional additives, such as alternative reducing agents (e.g., dithiothreitol) or stabilizers, to enhance performance for particular protein targets or experimental conditions. The instructions may include recommended mixing ratios, storage conditions, and safety precautions to ensure reliable preparation and consistent denaturation of hydrophobic proteins.

[0034] EXAMPLE

[0035] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.

[0036] Methods of Preparation of the Loading Buffer Composition at a 4X Concentration. A loading buffer composition was prepared by combining 5 mL of 18.2 M cm water with 5 mL of 1 M Tris-HCl (pH 6.8) in a tube and mixing by swirling. To this, 10 mL of -mercaptoethanol was added, followed by thorough mixing through inversion. Glycerol (25.2 g) was then introduced using a 25 mL pipette, and the solution was again mixed by inversion. Bromophenol blue (0.01 g) was added and mixing continued until partial dissolution. Sodium dodecyl sulfate (SDS; 6 g) was carefully incorporated, e.g., in two approximately equal portions, with partial mixing after each addition. The tube was tightly sealed, e.g., with the tube cap covered with parafilm, and placed on a rotator (~30 RPM) or orbital shaker for about 30 minutes to ensure complete dissolution of all components. Additional water (18.2 MQ cm) was added as needed to reach the final intended volume, and then the tube was placed again on the rotator (~30 RPM) or orbital shaker for about 5 minutes to ensure complete mixing. Once prepared, the solution was filtered through a nylon syringe filter that had been pre-wetted with water to ensure efficient passage of the loading buffer composition. Excess water from the filter was expelled by pushing air through before use. The prepared solution was then passed through the filter at a controlled rate (approximately 1-2 drops per second), with the initial diluted drops discarded, and the remaining solution aliquoted into 1.5 mL tubes. Each aliquot was securely capped immediately after filling, and the aliquots were stored at -80 °C for subsequent use.

[0037] Table 1 shows the stock concentrations and corresponding amounts of each component used in preparing the loading buffer at 4X concentration. This concentrated solution yields: 3% SDS 5% P-mercaptoethanol at IX.September 25, 2025

[0038] Methods of Use: To prepare a 1 x loading buffer solution, three volumes of sample are combined with one volume of the 4x loading buffer. For example, 30 pL of sample may be mixed with 10 pL of the 4x loading buffer. Protein samples prepared in this manner are typically heated at 95 °C for approximately 5 minutes prior to gel loading, although the optimal temperature and incubation time may vary depending on the protein. Following heating, samples should generally be loaded onto the gel within about 15 minutes to ensure effective denaturation and consistent electrophoretic performance.EMBODIMENTS

[0039] The following embodiments are intended to be illustrative only and not to be limiting in any way.

[0040] Embodiment 1: A loading buffer composition effective for denaturing hydrophobic proteins, the composition comprising: a) 10-15% wt / vol of a denaturing agent; and b) 15-25% vol / vol of a reducing agent; wherein the composition remains soluble.September 25, 2025

[0041] Embodiment 2: The composition of embodiment 1, wherein the denaturing agent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, or another ionic detergent suitable for protein denaturation. Embodiment 3: The composition of embodiment 1 or embodiment 2, wherein the composition comprises 12% wt / vol of the reducing agent. Embodiment 4: The composition of any one of embodiments 1-3, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT). Embodiment 5: The composition of any one of embodiments 1-4, wherein the reducing agent comprises 20% vol / vol.

[0042] Embodiment 6: The composition of any one of embodiments 1-5 further comprising one or a combination of water, a buffer, glycerol, or a tracking dye. Embodiment 7: The composition of embodiment 6, wherein the buffer is Tris-HCl (e.g., Tris HC1 pH 6.8). Embodiment 8: The composition of embodiment 6, wherein the tracking dye is bromophenol blue or xylene cyanol.

[0043] Embodiment 9: A method of denaturing a hydrophobic protein comprising: a) obtaining or having obtained a protein sample; b) diluting the protein sample in the loading buffer composition according to any one of embodiments 1-8; and c) heating the diluted protein sample for a period of time. Embodiment 10: A method of denaturing a hydrophobic protein comprising: a) obtaining or having obtained a protein sample; b) diluting the protein sample in the loading buffer composition comprising: i) 10-15% wt / vol of a denaturing agent; and ii) 15-25% vol / vol of a reducing agent; wherein the composition remain soluble; and c) heating the diluted protein sample for a period of time

[0044] Embodiment 11 : The method of embodiment 10, wherein the denaturing agent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, or another ionic detergent suitable for protein denaturation. Embodiment 12: The method of embodiment 10 or embodiment 11, wherein the composition comprises 12% wt / vol of the reducing agent. Embodiment 13: The method of any one of embodiments 10-12, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2- carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT), Embodiment 14: The method of any one of embodiments 10-13, wherein the reducing agent comprises 20% vol / vol.

[0045] Embodiment 15: The method of any one of embodiments 10-14, wherein the loading buffer composition further comprising one or a combination of water, a buffer, glycerol, or a tracking dye. Embodiment 16: The method of embodiment 15, wherein the buffer is Tris-HCl (e.g., Tris-HCl pH 6.8). Embodiment 17: The method of embodiment 15, wherein the tracking dye is bromophenol blue or xylene cyanol.September 25, 2025

[0046] Embodiment 18: The method of any one of embodiments 9-17, wherein the hydrophobic protein is a membrane protein. Embodiment 19: The method of any one of embodiments 9-18, wherein the protein sample is diluted in a 1 to 3 dilution; wherein 3 parts of the protein sample is diluted into one part of the loading buffer composition.

[0047] Embodiment 20: The method of any one of embodiments 9-19, wherein the diluted protein sample is heated to a temperature of about 90 to 100°C. Embodiment 21: The method of any one of embodiment 9-20, wherein the diluted protein sample is heated to a temperature of about 95°C. Embodiment 22: The method of any one of embodiments 9-21, wherein the diluted protein sample is heated for about 2 to 10 minutes. Embodiment 23: The method of any one of embodiments 9-22, wherein the diluted protein sample is heated for about 5 minutes.

[0048] Embodiment 24: The method of any one of embodiments 9-23, wherein the diluted protein sample is heated to a temperature of less than 90°C. Embodiment 25: A method of denaturing a hydrophobic protein at a temperature less than 90°C comprising; a) obtaining or having obtained a protein sample; b) diluting the protein sample in the loading buffer composition comprising: i) 10-15% wt / vol of a denaturing agent; and ii) 15-25% vol / vol of a reducing agent; wherein the composition remain soluble; and c) heating the diluted protein sample for a period of time at a temperature less than 90°C. Embodiment 26: The method of any one of embodiments 9- 25, wherein the diluted protein sample is heated for about 2 to 10 minutes.

[0049] Embodiment 27: The method of any one of embodiments 9-26, wherein the diluted protein sample is heated to a temperature of about 90°C. Embodiment 28: The method of any one of embodiments 9-27, wherein the diluted protein sample is heated to a temperature of about 85°C. Embodiment 29: The method of any one of embodiments 9-26, wherein the diluted protein sample is heated to a temperature of about 80°C.

[0050] Embodiment 30: A method of generating a loading buffer composition, the method comprising carrying out the following steps in sequence, concurrently, or in any order suitable for preparing the composition: a) adding water and buffer to a container; b) adding reducing agent; c) adding glycerol; d) adding an indicator; and e) adding a denaturing agent; wherein the container is mixed or inverted after each addition as desired.

[0051] Embodiment 31 : The method of embodiment 30, wherein the denaturing agent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, or another ionic detergent suitable for protein denaturation. Embodiment 32: The method of embodiment 30 or embodiment 31, wherein theSeptember 25, 2025 reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT). Embodiment 33: The method any one of embodiment 30-32, wherein the buffer is Tris-HCl (e.g., Tris-HCl pH 6.8) Embodiment 34: The method any one of embodiment 30-32, wherein the tracking dye is bromophenol blue or xylene cyanol.

[0052] Embodiment 35: A method of generating a loading buffer composition, the method comprising carrying out the following steps in sequence, concurrently, or in any order suitable for preparing the composition: a) adding water and Tris-HCl pH 6.8 to a container; b) adding beta- mercaptoethanol; c) adding glycerol; d) adding bromophenol blue; and e) adding sodium dodecyl sulfate (SDS); wherein the container is mixed or inverted after each addition as desired.

[0053] Embodiment 36: The method of any one of embodiments 30-35, wherein the composition comprises 10-15% wt / vol of the reducing agent. Embodiment 37: The method of any one of embodiments 30-36, wherein the composition comprises 12% wt / vol of the reducing agent. Embodiment 38: The method of any one of embodiments 30-37, wherein the reducing agent comprises 15-25% vol / voL Embodiment 39: The method of any one of embodiments 30-38, wherein the reducing agent comprises 20% vol / vol.

[0054] Embodiment 40: The method of any one of embodiments 30-39, further comprising filtering the loading buffer composition. Embodiment 41: The method of any one of embodiments 30-40, further comprising discarding the first several drops upon filtering the loading buffer composition. Embodiment 42: The method of any one of embodiments 30-41, further comprising storing the loading buffer composition at -80°C. Embodiment 43: The method of any one of embodiments 30-42, wherein the loading buffer composition is viable at -20°C for up to 6 months.

[0055] Embodiment 44: A system for denaturing hydrophobic proteins, comprising: a) a premixed solution comprising a denaturing agent, water, a buffer, glycerol, and a tracking dye, the premixed solution being formulated for combination with a reducing agent immediately prior to use; and b) instructions for preparing a working loading buffer composition by adding a reducing agent to the premixed solution. Embodiment 45: A system for denaturing hydrophobic proteins, comprising: a) a fully premixed loading buffer solution that includes a denaturing agent, water, a buffer, glycerol, a reducing agent, and a tracking dye, the solution being provided in a sealed container for immediate use; and b) instructions for denaturing a protein sample using the fully premixed solution. Embodiment 46: A system for denaturing hydrophobic proteins, comprising: a) separate containers each holding one or more of the following: a denaturing agent, a buffering agent, glycerol, a reducing agent, and a tracking dye; and b) instructions for preparing a loadingSeptember 25, 2025 buffer by combining the components in specified proportions.

[0056] Embodiment 47: The system of any one of embodiments 44-46, wherein the denaturing agent is sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, or another ionic detergent suitable for protein denaturation.

[0057] Embodiment 48: The system of any one of embodiments 44-47, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT). Embodiment 49: The system of any one of embodiments 44-48, wherein the buffer is Tris- HC1 (e.g., Tris-HCl pH 6.8). Embodiment 50: The system of any one of embodiments 44-49, wherein the tracking dye is bromophenol blue or xylene cyanol.

[0058] Embodiment 51: A method of detecting heat modifiability of a membrane protein, the method comprising; a) incubating a protein sample comprising the membrane protein in the loading buffer composition according to any one of embodiments 1-8 at a first incubation temperature, wherein at the first incubation temperature the membrane protein remains incompletely denatured, b) incubating the protein sample in the loading buffer composition at a second temperature, wherein at the second temperature the membrane protein is completely denatured; and c) an detecting a mobility shift of the protein on an SDS-PAGE gel, wherein the mobility shift is evidenced by migration of the membrane protein to a higher molecular weight band at the second temperature as compared to the first temperature, thereby identifying the protein as heat modifiable. Embodiment 52: The method of embodiment 51, wherein the first incubation temperature and the second incubation temperature are less than 90°C. Embodiment 53: The method of embodiment 51 or embodiment 52, wherein the first incubation temperature or the second incubation temperature is about 90°C. Embodiment 54: The method of embodiment 51 or embodiment 52, wherein the first incubation temperature or the second incubation temperature is about 85°C. Embodiment 55: The method of embodiment 51 or embodiment 52, wherein the first incubation temperature or the second incubation temperature is about 85°C. Embodiment 56: The method of any one of embodiments 51-55, wherein the first incubation temperature is lower than the second incubation temperature. Embodiment 57: The method of any one of embodiments 51- 56, wherein the protein sample is incubated at a first incubation temperature for about 2 to 10 minutes. Embodiment 58: The method of embodiment 51-57, wherein the protein sample is incubated at a first incubation temperature for about 5 minutes. Embodiment 59: The method of embodiment 51-58, wherein the protein sample is incubated at a second incubation temperature for about 2 to 10 minutes. Embodiment 60: The method of embodiment 51-59, wherein the proteinSeptember 25, 2025 sample is incubated at a second incubation temperature for about 5 minutes.

[0059] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0060] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

PCT / US25 / 48289 26 September 2025 (26.09.2025)SKSG 24.04 PCTSeptember 25, 2025WHAT IS CLAIMED IS1. A loading buffer composition effective for denaturing hydrophobic proteins, the composition comprising: a. 10-15% wt / vol of a denaturing agent; and b. 15-25% vol / vol of a reducing agent; wherein the denaturing agent and the reducing agent remain soluble in said composition.

2. The composition of claim 1, wherein the denaturing agent is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.

3. The composition of claim 1, wherein the composition comprises 12% wt / vol of the denaturing agent.

4. The composition of claim 1, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT).

5. The composition of claim 1, wherein the composition comprises 20% vol / vol of the reducing agent.

6. The composition of claim 1 further comprising one or a combination of water, a buffer, glycerol, or a tracking dye.

7. The composition of claim 6, wherein the buffer is Tris-HCl.

8. The composition of claim 6, wherein the tracking dye is bromophenol blue or xylene cyanol.

9. A method of denaturing a hydrophobic protein comprising: a. obtaining or having obtained a protein sample; b. diluting the protein sample in a loading buffer composition comprising: i. 10-15% wt / vol of a denaturing agent; and ii. 15-25% vol / vol of a reducing agent, wherein the denaturing agent and the reducing agent remain soluble in said composition; and c. heating the diluted protein sample for a period of time.PCT / US25 / 48289 26 September 2025 (26.09.2025)SKSG 24.04 PCTSeptember 25, 202510. The method of claim 9, wherein the denaturing agent is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.

11. The method of claim 9, wherein the composition comprises 12% wt / vol of the denaturing agent.

12. The method of claim 9, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT).

13. The method of claim 9, wherein the composition comprises 20% vol / vol of the reducing agent.

14. The method of claim 9 further comprising one or a combination of water, a buffer, glycerol, or a tracking dye.

15. The method of claim 14, wherein the buffer is Tris-HCl.

16. The method of claim 14, wherein the tracking dye is bromophenol blue or xylene cyanol.

17. The method of claim 9, wherein the hydrophobic protein is a membrane protein.

18. The method of claim 9, wherein the protein sample is diluted in a 1 to 3 dilution; wherein 3 parts of the protein sample is diluted into one part of the loading buffer composition.

19. The method of claim 9, wherein the diluted protein sample is heated to a temperature of about 90 to 100°C.

20. The method of claim 9, wherein the diluted protein sample is heated to a temperature of about 95°C.

21. The method of claim 9, wherein the diluted protein sample is heated for about 2 to 10 minutes.

22. The method of claim 9, wherein the diluted protein sample is heated for about 5 minutes.

23. The method of claim 9, wherein the diluted protein sample is heated to a temperature of less than 90°C.

24. The method of claim 9, wherein the diluted protein sample is heated to a temperature of about 90°C.PCT / US25 / 48289 26 September 2025 (26.09.2025)SKSG 24.04 PCTSeptember 25, 202525. The method of claim 9, wherein the diluted protein sample is heated to a temperature of about 85°C.

26. The method of claim 9, wherein the diluted protein sample is heated to a temperature of about 80°C.

27. A method of generating a loading buffer composition, the method comprising: a. adding water and buffer to a tube; b. adding reducing agent to the tube and inverting the tube at least once; c. adding glycerol to the tube and inverting the tube at least once; d. adding an indicator to the tube and inverting the tube at least once; and e. adding a denaturing agent to the tube and gently mixing the tube to avoid creating bubbles.

28. The method of claim 27, wherein the denaturing agent is sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate.

29. The method of claim 27, wherein the reducing agent is beta-mercaptoethanol (BME), Tris(2-carboxyethyl)phosphine (TCEP), or dithiothreitol (DTT).

30. The method of claim 27, wherein the buffer is Tris-HCl.

31. The method of claim 27, wherein the tracking dye is bromophenol blue or xylene cyanol.

32. The method of claim 27, wherein the composition comprises about 15-25% vol / vol of a reducing agent.

33. The method of claim 27, wherein the composition comprises about 20% vol / vol of the reducing agent.

34. The method of claim 27, wherein the composition comprises about 10-15% wt / vol of the denaturing agent.

35. The method of claim 27, wherein the composition comprises about 12% wt / vol of the denaturing agent.

36. The method of claim 27, further comprising filtering the loading buffer composition.

37. The method of claim 27, further comprising discarding the first several drops uponPCT / US25 / 48289 26 September 2025 (26.09.2025)SKSG 24.04 PCTSeptember 25, 2025 filtering the loading buffer composition.

38. The method of claim 27, further comprising storing the loading buffer composition at - 80°C.

39. The method of claim 27, wherein the loading buffer composition is viable at -20°C for up to 6 months.

40. A method of detecting heat modifiability of a membrane protein, the method comprising: a. incubating a protein sample comprising the membrane protein in the loading buffer composition according to claim 1 at a first incubation temperature, wherein at the first incubation temperature the membrane protein remains incompletely denatured, b. incubating the protein sample in the loading buffer composition according to claim 1 at a second temperature, wherein at the second temperature the membrane protein is completely denatured; and c. detecting a mobility shift of the protein on an SDS-PAGE gel, wherein the mobility shift is evidenced by migration of the membrane protein to a higher molecular weight band at the second temperature as compared to the first temperature, thereby identifying the protein as heat modifiable.

41. The method of claim 40, wherein the first incubation temperature and the second incubation temperature are less than 90°C.

42. The method of claim 40, wherein the first incubation temperature is lower than the second incubation temperature.

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