TITLE

FOAM TO DELIVER TEXTILE EFFECT ADDITIVES ON FIBROUS ARTICLES

FIELD

This disclosure relates to the field of treating fibrous articles using foams of amphiphilic proteins to improve the fibrous article treating process.

BACKGROUND

In a typical textile treatment process, textile effect additives are used to impart desirable properties or effects to a treated article. Fibrous articles such as textiles are usually treated by immersion of the fibrous article in a water bath containing the textile additives. The article becomes saturated with water in the process. Excess water must be removed from the fibrous article, and the fibrous article must then be dried to allow its further processing. The drying step can be time-consuming and energy inefficient.

The amount of water retained by the fibrous article (also known as

"wet pick up") is normally about 50 to 70 percent water based on the weight of the fibrous article. This large amount of water requires a tremendous amount of energy in the form of heat to dry the fibrous article. It has been estimated that the amount of energy required to remove the water and dry the fibrous article is many times greater than the amount of energy that is needed for curing the fibrous article to prepare it for additional finishes.

More recently, foams have been used to treat textiles and obtain uniform application of treatment compositions on porous articles while also reducing the amount of water used in the process.

However, it can still be problematic in a textile treatment process to have foam compositions available that have the adequate stability and penetration properties needed for treating a fibrous article. The

compositions disclosed herein are intended to address these issues in the treatment of fibrous articles. SUMMARY

In one aspect, this disclosure describes a foam composition comprising: (i) at least one amphiphilic protein; and (ii) at least one textile effect additive.

In another aspect, this disclosure describes a process for treating a fibrous article that is useful for producing a fibrous surface that is oil and water repellant, the process comprising the steps of:

a) producing a foam comprising: (i) at least one amphiphilic protein; and (ii) at least one textile effect additive;

b) contacting the foam with the fibrous surface in areas where oil and water repellency is desirable; and c) curing the foam on the fibrous surface, and optionally wherein the foam is produced by a process comprising the steps: i) providing a first aqueous solution comprising at least one textile effect additive; ii) providing a second aqueous solution comprising at least one amphiphilic protein; iii) contacting the first aqueous solution with the second aqueous solution to produce an aqueous mixture; iv) producing a foam phase and a liquid phase from the aqueous mixture; and v) allowing the foam phase to separate from the liquid phase to produce the foam comprising the additives of step (i) and the amphiphilic proteins of step (ii); wherein the fibrous surface is treated in the absence of any wetting or thickening agents, and wherein the fibrous surface has a water and oil repellency of greater than 1 after it is treated.

DESCRIPTION OF THE FIGURES Figures 1A and 1 B are screening electron microscope (SEM) images of a piece of cotton that is covered with a mixture of class II hydrophobin and fluoro additive Capstone® TC . Image (a) is the uncoated control, image (d) is the fabric with 1 .67% fluoro additive, image (g) is the fabric with 3.3% fluoro additive, image (j) is the fabric with 6.67% fluoro additive. Images (b, e, h and k) show the 1000 magnification of each of the coated fabrics. Images (c, f, i and I) show the 3000 magnification of each of the coated fabrics.

DETAILED DESCRIPTION

Described herein is a foam composition comprising at least one amphiphilic protein and at least one textile effect additive to treat fibrous articles. The at least one textile effect additive in the foam imparts oil and/or water repellency to the fibrous article while the at least one amphiphilic protein in the foam further reduces wet pick up of the treated fibrous article.

The term "foam" refers to a mixture of gas and liquid with the foam structure consisting of gas pockets trapped in a network of thin liquid films. In the instant disclosure the foam comprises at least one amphiphilic protein and at least one textile effect additive. Upon standing, a foam phase and a liquid phase, is produced.

The term "amphiphilic protein" refers to protein structures that possess both hydrophilic and hydrophobic domains.

The term "textile effect additives" as the term is used herein refers to chemical additives used to impart specific properties to a textile that has been treated with said additives.

The term "fibrous articles" refers to natural or synthetic fibrous articles that are flexible woven or non-woven materials consisting of a network of natural or artificial fibers often referred to as thread or yarn. Fibrous articles are formed by weaving, knitting, crocheting, knotting, or pressing fibers together. Fibrous articles can be composed of fibers of cotton, rayon, silk, wool, polyester, polypropylene, polyolefins, nylon, and aramids such as "NOMEX" and "KEVLAR".

Amphiphilic proteins

Amphiphilic proteins suitable for use as described herein can be in the form of oligomeric mixtures comprising, typically, not more than several hundred monomeric protein units. The number of monomeric units of a suitable protein is practically limited only by the stability of the oligomer in the mixture.

Amphiphilic proteins useful in producing the foams described herein are, for example, hydrophobins, bovine serum albumin, natural and synthetic silk proteins which have been derived from the natural silk and have been heterologously expressed in suitable prokaryotic or eukaryotic expression systems using genetic engineering processes; wheat proteins; protein hydrolysate, amphiphilic plant-derived or animal-derived proteins; genetically or naturally modified amphiphilic plant-derived or animal- derived proteins; soy wheat protein; or combinations of any of these.

These amphiphilic proteins can be in their natural conformations, or subjected to denaturation treatments to enhance their amphiphilicity.

Other amphiphilic proteins may be suitable for use herein, and this listing is not to be considered all inclusive.

One of the classes of amphiphilic proteins are naturally produced hydrophobins. In addition hydrophobin derivatives, or hydrophobin-like materials comprising chemically modified or genetically modified hydrophobins, can also be used in the in the practices described and claimed herein. Examples of such hydrophobin modifications include glycosylation, or acetylation or by chemical cross-linking for example with glutaraldehyde, or by cross-linking with a polysaccharide such as heparin. Hydrophobin-like proteins have the self-assembly property of the original hydrophobin at hydrophilic or hydrophobic interfaces into amphipathic coatings. For the purposes of the present use in a foam composition, hydrophobins can be unmodified (natural), genetically modified or chemically modified hydrophobins.

Hydrophobins are known and described, for example in the

International Publications WO2012/142557 A1 , and WO2012/135433 which are hereby incorporated by reference as if fully set out herein.

Hydrophobins are divided into two classes, class I and class II, based on their structural and solubility differences in water. In the processes described in the instant disclosure, both class I and class II hydrophobins are suitable for use in preparing the foams described herein. Further, in the instant disclosure, at least one class II hydrophobin can be used.

Class II hydrophobins possess an almost globular structure that is highly cross-linked by four internal disulfide bonds. One face of the protein shows nearly exclusively aliphatic hydrophobic residues. The rest of the protein shows typical hydrogen-bonding and charged residues which make the molecule amphiphilic. The amphiphilic structure of class II hydrophobin proteins explains their localization on interfaces between polar and nonpolar substances such as the air-water interface and on hydrophobic surfaces in water. Class II hydrophobins exist in water as dimers, tetramers and other higher oligomers, with their hydrophobic regions concealed between each pair of hydrophobins. Thus, their solubility in water is greatly enhanced due to the exposure of the hydrophilic surfaces to the aqueous environment. The degree of oligomerization of hydrophobins in water depends on the pH and ionic strength of the water, and the concentration of the hydrophobins in the water.

At least one hydrophobin can be present as an initial component of the composition disclosed herein. Alternatively, the at least one hydrophobin can be generated in situ in the composition (for example, by in situ hydrolysis of a hydrophobin fusion protein).

In the instant disclosure, class I or class II hydrophobins, or any mixture of them can be used. In one embodiment, at least one amphiphilic protein is a class II hydrophobin. In one embodiment, at least one amphiphilic protein is bovine serum albumin. Composition of the foam

The composition of the foam disclosed herein comprises: a) at least one textile effect additive and, b) at least one amphiphilic protein.

Amphiphilic proteins useful in preparing the foams of the instant disclosure can be in the form of a solution, a dispersion, or a suspension.

The foam composition comprises at least one amphiphilic protein in an aqueous solution. This aqueous solution can be water or a mixture of water and one or more organic solvents. The identity of the organic solvent that can be mixed with the at least one amphiphilic protein depends on the amphiphilic protein used and the intended application for the coated article. Examples of suitable organic solvents useful in the practice of the instant are: acetaldehyde, acetone, acetonitrile, butanol, diethanolamine, dimethylsulfoxide, dioxane, ethanol, ethylamine, ethylene glycol, methanol, propanol, and tetrahydrofuran, for example.

In some embodiments, the amphiphilic protein has a concentration of from about 0.005 wt% to about 0.1 wt% of the foam. In another embodiment, the amphiphilic protein has a concentration of from about 0.01 wt% to about 0.1 wt% of the foam.

In another embodiment, at least one amphiphilic protein is a class II hydrophobin and wherein the class II hydrophobin has a concentration of from about 0.005 wt% to about 0.1 wt% of the foam. In another

embodiment, at least one amphiphilic protein is a class II hydrophobin and wherein the class II hydrophobin has a concentration of from about 0.01 wt% to about 0.1 wt% of the foam.

The upper concentration limit of the organic solvent in the aqueous solution comprising the amphiphilic protein can depend on the effect of the solvent on the formulation stability and foaming capability and the desired application. For example, ethanol can be used at concentrations up to about 50 weight% of the total solution for some applications. However, for most applications an ethanol concentration of from about 10 weight% to about 20 weight% of the total solution would be suitable.

Fibrous articles coated with foam compositions disclosed herein possess water repellency, as determined by the Water Repellency Test described herein below, in the range of from about 1 - 2 when the foam does not comprise the at least one textile effect additive, and greater than 4 when the at least one textile effect additive is present in the foam in addition to the amphiphilic protein.

a) The textile effect additives suitable for use in the foams described herein can be used alone or in combination with other textile effect additives and other optional additives. For example, it can be desirable to treat a fabric or a textile article with additives that can impart water repellency, oil repellency, dirt repellency, resistance to static electricity build-up.

Additives that can be suitable for delivering such properties to a fibrous article are, for example, copolymers that can either be fluorinated copolymers or non-fluorinated copolymers.

Copolymers suitable for use in foams described herein can be various (meth)acrylate copolymers. The term "(meth)acrylate" is used herein to mean either methacrylate, acrylate or a mixture of the two.

Copolymers that contain at least one monomer containing fluorine are referred to herein as "F-copolymers". For example fluoro(meth)acrylates such as those commercially available from

E. I. du Pont de Nemours and Company, Wilmington, DE as Capstone® is an F-copolymer suitable for use herein.

Such additives not specifically named herein that are conventional and known in the treatment of textiles should be considered as useful herein.

In one embodiment, the textile effect additives comprise a fluorinated copolymer (F-copolymer), which can be included in an amount of from about 5 to about 200 g F-copolymer per liter of treatment solution. The F-copolymers useful herein can be methacrylate polymers based on: C ₆ Fi3-CH2CH ₂ OC(O)C(CH3)=CH2.

The F-copolymers useful herein have been fully described in US Patent Application Serial 14/033542, which is hereby incorporated by reference as if fully set out herein.

Suitable F-copolymers useful herein can be blended with a variety of known treating agents used for fibrous articles. Examples of commercially available such treating agents are ZONYL® and

CAPSTONE® TC available from E. I. du Pont de Nemours and Company, Wilmington, DE; OLEOPHOBOL® from Ciba Specialty Chemicals,

Langweid, Germany; ASAHI GARD from Asahi Glass Company, Ltd., Tokyo, Japan; UNIDYNE™ from Daikin America, Inc., Orangeburg, NY; SCOTCH GARD® from 3M Company, St. Paul, MN; and NANO TEX™ from Nanotex, Emeryville, CA.

Various specialized monomers can be incorporated into the F- copolymer solution, these polymers are generally marketed as aqueous solutions for easy application to the fibrous articles. Since each ingredient can impart some potentially undesirable properties in addition to its desirable ones, the specific combination is directed to the desired use. F- copolymers of the type useful herein are commercially available and are generally marketed as aqueous solutions for easy application to the fibrous articles. Thus, as used herein, the term "aqueous solution" refers to any solution liquid, emulsion, suspension, colloidal and mixtures thereof.

Non-fluorinated polyurethanes are also suitable for use herein as copolymers. Polyurethanes are prepared by reacting: (a) at least one isocyanate group-containing compound selected from isocyanate, polyisocyanate with (b) at least one isocyanate-reactive compound selected from formula (below):

wherein each R is independently a -C(O)R ¹ and wherein each R ¹ is

Suitable non-fluorinated polyurethane polymers for application herein have been disclosed in the US Patent Application Serial 14/226892, which is hereby incorporated as if fully set out herein. Additional non- fluorinated polyurethane polymers are commercially available. Non-fluorinated polyurethane polymers that are "bio-derived" can be suitable for use herein.

Fibrous articles coated with foam compositions disclosed herein can possess water and oil repellency in the range of from about 1 to about 2 when the foam does not comprise the at least one textile effect additive, and greater than about 4 when the at least one textile effect additive is present in the foam in addition to the amphiphilic protein.

In some embodiments, the foam composition comprises a wetting or thickening agent. In some embodiments, a water and oil repellency of greater than 1 is achieved without the use of a wetting or thickening agent. The water repellency and oil repellency was measured according to the DuPont Technical Laboratory Method as outlined in the TEFLON ® Global Specifications and Quality Control Tests information packet or AATCC standard Test Method No. 193-2004.

Foam compositions comprising F-copolymers can provide both oil and water repellency. Foam compositions comprising non-F-copolymers can provide water repellency when used as disclosed herein.

Production of the foam

For the production of the foam according to the instant disclosure, a first aqueous solution comprising at least one textile effect additive can be contacted with a second aqueous solution comprising at least one amphiphilic protein to provide an aqueous mixture of these solutions.

Thus, "aqueous mixture" as used herein comprises an aqueous mixture of one or more amphiphilic proteins and one or more textile effect additives.

The term "contact or contacting", as used herein, refers to adding, mixing, pouring, dumping of the at least one copolymer in an aqueous solution with the aqueous solution of at least one amphiphilic protein.

Optionally, the at least one amphiphilic protein can be in the powder form before contacting the first aqueous solution of the at least one textile effect additive. Alternatively, the second aqueous solution comprising the at least one amphiphilic protein can be foamed to provide the foam and then be contacted with the first aqueous solution comprising the at least one textile effect additive. The aqueous solution of the textile effect additives can further comprise additional components such as additional treating agents or finishes to achieve additional surface effects, or additives commonly used with such agents or finishes. Such additional components comprise compounds or compositions that provide surface effects such as no iron, easy to iron, shrinkage control, wrinkle free, permanent press, moisture control, softness, strength, anti-slip, anti-static, anti-snag, anti-pill, stain repellency, stain release, soil repellency, soil release, water repellency, oil repellency, odor control, antimicrobial, sun protection, and similar effects. One or more such treating agents or finishes can be combined with the blended composition and applied to the fibrous substrate. Other additives commonly used with such treating agents or finishes may also be present such as surfactants, pH adjusters, cross linkers, wetting agents, and other additives known by those skilled in the art.

Following contacting the first aqueous solution of the at least one textile effect additive, and the second aqueous solution of the at least one amphiphilic protein an aqueous mixture is produced. The concentration of the amphiphilic protein in the aqueous mixture can be from about 0.005 weight% to about 25 weight% based on the weight of the final solution. Alternatively, the concentration of the amphiphilic protein in the aqueous mixture can be from about 0.01 weight% to about 0.1 weight% based on the weight of the mixture of the first and second solutions.

The foam suitable for the instant disclosure can be produced using various commercially available foam generating devices. One type of foam generator uses a mechanical agitator capable of quantitatively mixing a gas and a liquid composition containing hydrophobin. Another type of foam generator contains a proportioner to mix gas and pressurized liquid containing one or more amphiphilic proteins in proportions appropriate for forming a foam useful in the practice of the instant disclosure. Any type of instrument known to practitioners of the relevant art can be used to produce the foam useful for the instant disclosure.

Production of the foam results in formation of a foam phase and a liquid phase. The water content of the foam can decrease through natural foam drainage. In this method water can flow out of the foam due to gravity. In the practice of the instant disclosure, the foam comprising at least one amphiphilic protein and at least one textile effect additive can be allowed to stand for from about 1 , to about 30 minutes to allow water drainage. The drainage process can be facilitated using gravity-based methods such as centrifugation, or by pressure difference such as vacuum suction or capillary suction.

The presence of the at least one amphiphilic protein in the foam prevents collapsing of the foam even at low water content. The water content of a foam, as used herein, can be quantified based on the foam density. The density of the foam can be measured using methods well- known in the relevant art. As used herein, the term "foam" refers to a foam with a density in the range of from about 0.001 gram(s) per milliliter to about 0.5 gram(s) per milliliter. Alternatively, the density of the foam can be in the range of from about 0.005 to about 0.1 gram(s) per milliliter.

Further, the density of the foam can be in the range of from about 0.01 to about 0.05 gram(s) per milliliter.

The foams of the instant disclosure permit treatment of fibrous articles with any number of textile effect additives to impart a particular property or treatment thereto. The disclosed foam compositions can comprise flame-retarding reagents, water-proofing or water-repeilant reagents, mildew proofing reagents, bacteriostats, antistats permanent press or wash and wear compositions, softeners, lubricants, hand builders, dyes, pigments, sizes, whitening agents, fluorescent brighteners, bleaches, binders for non-woven fabrics, latexes, scouring agents, thermal or radiation curable monomers or oligomers or polymers, soil or stain release agents, or any other additives known to be used in the treatment of fibrous articles or papers.

The foam composition is not limited to any particular functional or treating agent or combination of agents. Illustrative of typical functional agents include, but are not limited to, dimethyioidihydroxy- ethylene urea, dimethylolethylene urea, dimethylolpropylene urea, urea formaldehyde resins, dimethyiol urons, the methyiolated melamines, methyloiated triazones; the methyiolated carbamates such as the ethyl or methoxyethyl or isopropyl or butyl carbamates; the epoxides such as vinyl cyclohexene dioxide, 2,3-dia!lyoxy-1 ,4-dioxane, 2,3-bis(2,3~epoxypropoxy)-1 ,4-dioxane, the diglycidyl ether of bisphenol-A, bis(3,4-epoxybutyl)ether; flame- proofing agents such as tetrakis hydroxymethyl phosphonium chloride, polyvinyl chloride latexes, (N-hydroxymethyl-3-dimethyi

phospono)propionamide; water-proofing or water repelianf agents such as aluminum formate, sodium formoacetate, methylene bis-stearamide;

mildew proofing and bacteriostat agents such as copper-8-quinolinoiate, dihydroxydichlorodiphenylmethane, zinc salts of dimethyldithiocarbamic acid, dihydroxymethyl undecyienamide; latexes such as polyvinyl acetate latexes, acrylic latexes, styrene-butadiene latexes; softeners such as emuisifiabie polyethylene, dimethyl stearate ammonium salts; lubricants such as butyl stearate, diethyiene glycol stearate, polyethylene glycol, polypropylene glycol; hand builders such as polyvinyl acetate latexes, acrylic latexes, styrene-butadiene latexes; dyes and pigments such as Acid Blue 25 (Color Index 62055), Acid Red 151 (Color Index 26900),

Direct Red 39 (Color Index 23630), Dispersed Red 4 (Color Index 60755), Phthalocyanine Blue 15 (Color Index 74160); polyvinyl alcohol, corn starch; whitening agents such as 4-methyl-7-diethyi- aminocoumarine; bleaches such as sodium hypochlorite, chlorine, hydrogen peroxide, dichlorodimethyl hydantoin, sodium perborate; binders for non-woven fibrous articles such as ethylene-vinyi acetate emulsion polymer, acrylic emulsion polymer, vinyl-acrylic copolymer; scouring agents such as sodium lauryl sulfate, triethanolamine lauryl sulfate, sodium N-methyl-N- oleoyltaurate, primary and secondary alcohol ethoxylates; radiation curable monomers and oligomers such as 2-hydroxyethyi acryiate, neopentyi glycol diacry!ate, pentaerythritol triacryiate, isodecyl acryiate, acrylated epoxidized soybean or linseed oil; antistatic agents such as ethoxylated steary! amines; soil or stain release agents such as acrylic polymers and fluorocarbon emulsions.

The foam composition of the instant disclosure can further comprise additional components such as surfactants, e.g., anionic, non-ionic, amphoteric and/or cationic surfactants, acids or bases; non-ionic polymers; polyelectrolytes; buffer systems; inorganic particles, such as S1O2 or silicates dyestuff; or biocide; UV absorber; and free-radical trapping. Such additional compounds can be selected depending on the application or intended use of the treated article obtained by the process described herein, or otherwise based on desirable properties or

performance enhancements that can result from use of such additional compounds. For example polar organic solvents that are miscible with water such as ethanol, acetone, dioxane, dimethylformamide and dimethylsulfoxide and others can be used.

The foam composition of the instant disclosure can further comprise additional components typically observed in normal textile treatment solutions to aid in runnability and durability, as is well known in the art. For example, when synthetic fibrous articles are treated, a wetting agent can be used, such as ALKANOL® 61 12 available from E. I. du Pont

de Nemours and Company, Wilmington, DE. Wetting agents are compounds that lower the surface tension (or interfacial tension) between two liquids or between a liquid and a solid. Wettings can act as detergents, surfactants, emulsifiers, foaming agents, and dispersants. As a further example, when cotton or cotton-blended fibrous articles are treated, a wrinkle-resistant resin can be used such as PERMAFRESH EFC available from Omnova Solutions, Chester, SC. As another example, for treatment of nonwoven fibrous articles a wax extender such as FREEPEL®

1225WR, available from Omnova Solutions Chester, SC can be used. An antistat such as ZELEC® KC, available from Stepan, Northfield, IL, or a wetting agent, such as hexanol, can also be suitable.

An important requirement of the selected functional or treating agent, to be added either to the first aqueous solution or the second aqueous solution, is that it does not interfere with the foam generation, nor with the foam properties to the extent that the foam composition could not be properly conveyed to the applicator nozzle or that the foam could not be properly applied to the article in a manner and form that it would rapidly break and uniformly penetrate the article.

Density of the foam is substantially equal to the amount of water in the foam. The freshly prepared amphiphilic protein foam can have high water content for example, higher than about 0.5 grams protein per milliliters, which is not a problem for the treating process but can be undesirable for the energy/water saving purposes.

The concentration of the at least one amphiphilic protein (e.g., Class II hydrophobin) in the second aqueous solution can be from about 0.005 weight% to about 25 weight% based on the weight of the solution. Alternatively, the concentration of the at least one amphiphilic protein (e.g., Class II hydrophobin) in the second aqueous solution can be from about 0.01 weight% to about 0.1 weight% based on the weight of the solution.

The concentration of the at least one amphiphilic protein (e.g., Class II hydrophobin) in the foam can be determined by the person skilled in the art using well known methods for any specific application. For example, the concentration of the at least one class II hydrophobin in the foam can be from about 0.005 weight % to about 0.1 weight %.

Alternatively, this concentration can be from about 0.01 weight % to about 0.02 weight %. The weight % herein is the weight of the hydrophobin as a percentage of the weight of the foam (M _H FB / M _f0 am)-

Treating the fibrous article

There are various methods suitable for treating the surface of a fibrous article with the foam produced using process described in the instant disclosure. As used herein, the term "treating or treatment" refers to applying, coating, covering, dosing, layering, contacting, spraying, dipping, or any other well-known methods in the art. Herein, treating a fibrous article refers to applying at least one layer of foam comprising at least one amphiphilic protein and at least one textile effect additive to the surface of the article.

In an embodiment of the instant disclosure at least one surface, or a portion of one surface of the fibrous article, comprising fibers, can be coated with the disclosed foam. The coating can be applied as one or more layers. As used herein, the term "coating" refers to partially covering at least one surface of the fibrous article. The one or more layers applied to the surface of the fibrous article can cover at least about 10%, or alternatively at least about 25% and or alternatively at least about 50% of the entire surface of the at least one surface of the fibrous article according to the instant disclosure. The degree of coverage of the fibrous article following treating can be determined by conventional methods such as microscopic methods well known in the art.

It is not required that the solution of the amphiphilic protein and the solution of the textile effect additives are contacted at the same

temperature. Alternatively, both solutions can be at the same

temperature. The aqueous solutions of the instant disclosure can be at the ambient temperature of from about 14 °C to about 50 °C. Alternatively, these solutions can be at temperatures from about 22 °C to about 35 °C. In embodiments of the instant disclosure, the textile effect additives

Capstone® TC or polyurethane and the hydrophobin II aqueous solutions are at ambient temperature (e.g., 15 - 35 °C).

For the practice of the instant disclosure, at least one textile additive or mixtures thereof can be used in all embodiments.

The concentration of hydrophobin II on the fibrous article surface of the instant disclosure can be estimated using a variety of methods well known in the art such as time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS).

In the practice of the instant disclosure, the concentration of the at least one amphiphilic protein and the at least one textile effect additive, in the final aqueous solution, can be determined based on the nature of the amphiphilic protein and the nature of the textile effect additive. The textile effect additive can be used from about 3 weight% up to about 25 weight% of the final solution in the instant disclosure. Treating of the surface of the fibrous article with the foam of the instant disclosure can be performed using various tools such as a roller or a brush. In embodiments of the instant disclosure, a standard paint roller and a foam brush are used. The foam of this disclosure can be used to apply a single treatment, or a multiplicity of treatments. A plurality of foaming and application systems can be used. The fibrous article can be treated with foam followed by subsequent drying or drying and curing. Further, since the amount of the foam added on to the article is generally below the water retention capacity of the article, the article can be rolled up without drying and stored or transferred to another location for subsequent use or treatment. The article which has been treated with the foam can be, but need not be, dry. The ability to apply a desired quantity of the foam is independent of the initial state of dryness of the article, provided the article is not completely saturated. This is a unique, unexpected, unobvious, and desirable feature of this process.

The multiple application of two or more foam compositions in succession, using separate textile effect additives, and applicator nozzles for each, with or without intermediate drying, curing or take-up steps is possible with the foams. This multiple application procedure is of particular advantage when the separate treatments or the functional reactants present in the foams are not compatible with each other or are too reactive with one another to be present in a single formulation or foam disclosed herein.

Following treating of the fibrous article with the foam disclosed herein comprising the at least one textile effect additive, the excess water should be removed from the fibrous article, for example, by squeeze rolls. The treated fibrous article can then be dried and cured by heating. The curing condition depends entirely on the curing requirements of the textile effect additive (i.e. Capstone® TC or polyurethane). Curing can be performed using a forced air oven, impact IR, drum dryer, tumble dryer and other equipment well known in the relevant art. Such curing enhances oil and water repellency and durability of the repellency. While these curing conditions are typical, some commercial apparatus may operate outside these ranges because of its specific design features. Additional curing methods can include thermal treating of the article or exposure to non-ionizing or ionizing radiation. In any instance any of the known thermal or radiation treatments pertinent to the particular formulation and article can be employed. Thus, for drying or thermal cure one can use infrared lamps, hot gases, ovens, heated rollers, or similar conventional heating means. For radiation curing one can use ultra-violet radiation, gamma radiation, electron beam radiation, or similar

conventional means. All these methods are well known in the relevant art.

In the instant disclosure the curing step can be performed at suitable temperatures from about 50 °C to about 180 °C for at least about 30 seconds. In an embodiment of the instant disclosure, the treated article is cured at up to about 165 °C for up to about 5 minutes.

Suitable fiber for the instant disclosure include paper, nonwovens, leather, fibers, textiles, fabrics, fibrous blend articles, or combinations thereof. By "fibrous blend articles" is meant fibrous articles made of two or more types of fibers. Typically these blends are a combination of at least one natural fiber and at least one synthetic fiber, but also can be a blend of two or more natural fibers or of two or more synthetic fibers. Such fibrous articles can be treated textile effect additives composition comprising a F-copolymer such as a poly(meth)acrylate, or a non- fluorinated polymer such as a polyurethane or poly(meth)acrylate or mixtures thereof. Alternatively, the textile effect additive for a fibrous article can contain F-copolymer such as a poly(meth)acrylate, and a non- fluorinated polymer such as a polyurethane and or any mixtures thereof.

The treated fibrous articles of the instant disclosure are useful in a variety of applications such as for textiles, clothing, uniforms, protective garments, furnishings, and the like. The articles of the instant disclosure are advantageous in that when blended with fluorinated polymer textile effect additive and the foam, they give highly durable, low yellowing repellent finishes over a wide range of fibrous articles while using a reduced level of the fluorinated polymer. Thus, the compositions disclosed herein are made at lower materials cost than the currently available acrylate copolymers.

Additionally, one of the unexpected advantages of the instant disclosure is that, unlike other foaming agents, presence of a thickening agent, e.g., xanthan gum, guar gum, methylcellulose, hydroxyethyl cellulose, polysaccharides or alginates is not needed to provide stable foams.

In some embodiments, the treated fibrous article comprises a fiberous article with at least one surface, comprising a coating of the foam composition located adjacent to the fiber surface.

In some embodiments, the treated fibrous article comprises a fibrous article with at least one surface, comprising a coating of the foam composition located adjacent to the fiber surface and wherein the at least one amphiphilic protein comprises a class II hydrophobin and wherein the concentration of the class II hydrophobin in the coating is about 0.1 milligram per gram of treated fiber.

In another embodiment, the treated fibrous article comprises fibrous article and one or more coatings of the foam composition on at least one surface of the fibrous article.

In another embodiment, the treated fibrous article comprises fibrous article and one or more coatings of the foam composition on at least one surface of the fibrous article and wherein the at least one amphiphilic protein is a class II hydrophobin and wherein the concentration of the at least one class II hydrophobin is from 0.005 wt% to 0.1 wt% based on the weight of the foam

One embodiment of the present disclosure is a process for treating a fibrous article. In one embodiment, a process for treating a fibrous article that is useful for producing a fibrous surface that is oil and water repellant comprises the steps of:

a) producing a foam comprising: (i) at least one amphiphilic protein; and (ii) at least one textile effect additive;

b) contacting at least one surface of the fibrous surface with the foam; and

c) curing the foam on the fibrous surface.

In some embodiments, step b is conducted in the absence of a wetting or thickening agent, and wherein the fibrous article has a water and oil repellency of greater than about 1 . In some embodiments, the fibrous article surface is cured at about 165°C for about 5 minutes.

In another embodiment, the process for treating a fibrous article further comprises the steps for producing the foam, comprising:

i) providing a first aqueous solution comprising at least one textile effect additive;

ii) providing a second aqueous solution comprising at least one amphiphilic protein;

iii) contacting the first aqueous solution with the second aqueous solution to produce an aqueous mixture; iv) producing a foam phase and a liquid phase from the aqueous mixture; and

v) allowing the foam phase to separate from the liquid phase to produce the foam comprising the additive of step (i) and the amphiphilic protein of step (ii);

Another embodiment of the present disclosure is treating a fibrous article that is useful for producing a fibrous surface that is oil and water repellant, the process comprising the steps of:

a) producing a foam comprising: (i) at least one amphiphilic protein; and (ii) at least one textile effect additive;

b) contacting at least one surface of the fibrous article with the foam; and

c) curing the foam on the fibrous article.

In some embodiments, step b is of a wetting or thickening agent, and wherein the fibrous article has a water and oil repellency of greater than about 1 .

In another embodiment, the process for treating a fibrous article further comprises the steps of:

i) providing a first aqueous solution comprising at least one textile effect additive;

ii) providing a second aqueous solution comprising at least one amphiphilic protein;

iii) contacting the first aqueous solution with the second aqueous solution to produce an aqueous mixture;

iv) producing a foam phase and a liquid phase from the aqueous mixture; and conducted in the absence with a density from 0.001 gram(s) per milliliter to 0.5 gram(s) per milliliter.

Another embodiment of the present disclosure is treating a fibrous article that is useful for producing a fibrous surface that is oil and water repellant, the process comprising the steps of:

i) providing a first aqueous solution comprising at least one textile effect additive; ii) providing a second aqueous solution comprising at least one amphiphilic protein;

iii) contacting the first aqueous solution with the second aqueous solution to produce an aqueous mixture;

iv) producing a foam phase and a liquid phase from the aqueous mixture; and decreasing the water content to produce a foam with a density from 0.001 gram(s) per milliliter to 0.5 gram(s) per milliliter, v) producing a foam comprising: (i) at least one amphiphilic protein; and (ii) at least one textile effect additive;

vi) contacting at least one surface of the fibrous article with the foam; and

vii) curing the foam on the fibrous article.

In some embodiments, the curing step is performed at temperatures from 50°C to 180°C for at least 30 seconds. In another embodiment, the curing step is performed at temperatures from 165 °C for 5 minutes.

In one embodiment, the concentration of the at least one

amphiphilic protein in the second aqueous solution is from 0.005 weight % to 25 weight % based on the weight of the second aqueous solution. In another embodiment, the concentration of the at least one amphiphilic protein in the second aqueous solution is from 0.01 weight % to 0.1 weight % based on the weight of the second aqueous solution.

EXAMPLES

The disclosure is further described and illustrated in, but not limited to, the following specific embodiments.

The following abbreviations are used in the Examples:

"g" is gram(s); "wt%" is weight percent; "μηη" is micrometer(s); and "nm" is nanometer(s); "cm" is centimeter(s). "g/L" is gram(s) per liter(s); "AATCC" is American Association of Textile Chemists and colorists; "μ" is

micrometer; "g/mL" is gram(s) per milliliter(s); "μΙ" is microliter(s); "Vol%" is volume percent; "mlM/m" is milli Newton per meter; "DTT" is dithiothreitol. Scanning Electron Microscopy (SEM)

Hitachi model s-4700 Cold Field Emission Gun Scanning Electron

Microscope (FEGSEM, Hitachi, Japan) was used to monitor the deposition of Capstone® TC on cotton fibers. The electron beam was set at 5kv for optimal surface topography and contrast. A < 5 nm carbon coating was applied to improve sample conductivity. In sample preparation, a carbon coating (< 5nm) was applied using a SPI-module sputter coater (Structure Probe, Inc., West Chester, PA, United States) to improve sample conductivity under the electron beam.

Time of flight secondary ion mass spectrometry (TOF-SIMS)

TOF-SIMS can be used for qualitative detection of hydrophobin on the painted panels. TOF-SIMS uses a focused primary ion beam to sputter the fabric surface and analyze the ejected secondary ions in a mass spectrometer to determine the elemental or molecular composition. The presence of hydrophobin residue can be confirmed based on the detection profile of H, C, O, N elements in comparison with a hydrophobin standard. X-ray photoelectron spectroscopy (XPS)

To obtain information about the concentration of carbon, nitrogen, sulfur, fluorine and oxygen on the hydrophobin coated substrates, which would allow determination of the extent of hydrophobin coating on each substrate, coated substrates is analyzed with an X-ray photoelectron spectroscopy (XPS). XPS is a surface-sensitive quantitative spectroscopic technique that measures the binding energy and the number of

photoelectrons to infer the surface composition. The coated surface is first examined by a broad survey scan to determine what elements are present on the surface. High resolution spectra can be then acquired to determine the chemical states of the detected elements and their atomic

concentrations. Bicinchoninic acid assay (BCA assay) for protein determination

Protein concentration is determined using BCA assay and bovine serum albumin (BSA), as an standard, can be obtained from Thermoscientific, Waltham, MA Assay is performed as recommended by the manufacturer.

Determination of wet pick up

To determine the wet pick up of a fibrous article, the weight of the fibrous article before (MO) and after dip and squeeze (M1 ) were recorded and the wet pick-up was calculated as the weight gain as a percentage of base fabric (M1 -M0)/M0.

Test Method - Water Repellency

The water repellency of a treated article was measured according to the DuPont Technical Laboratory Method as outlined in the TEFLON ® Global Specifications and Quality Control Tests information packet or AATCC standard Test Method No. 193-2004. The test determines the resistance of a treated article to wetting by aqueous liquids. Drops of water-alcohol mixtures of varying surface tensions are placed on the fibrous article and the extent of surface wetting is determined visually. The test provides a rough index of aqueous stain resistance. The higher the water repellency rating, the better the resistance of a finished article to staining by water- based substances. The composition of standard test liquids is shown in the following table.

Table 1

Standard Test Liquids for Water Repellency

Test Method - Oil Repellency The treated fibrous article articles were tested for oil repellency by a modification of AATCC standard Test Method No. 1 18, conducted as follows. A fibrous article, treated with an aqueous dispersion of polymer as previously described, is conditioned for a minimum of 2 hours at 23°C + 20% relative humidity and 65°C + 10% relative humidity. A series of organic liquids, identified below in Table I, are then applied drop wise to the fibrous article samples. Beginning with the lowest numbered test liquid (Repellency Rating No. 1 ), one drop (approximately 5 mm in diameter or 0.05 mL volume) is placed on each of three locations at least 5 mm apart. The drops are observed for 30 seconds. If, at the end of this period, two of the three drops are still spherical in shape with no wicking around the drops, three drops of the next highest numbered liquid are placed on adjacent sites and similarly observed for 30 seconds. The procedure is continued until one of the test liquids results in two of the three drops failing to remain spherical to hemispherical, or wetting or wicking occurs. The oil repellency rating of the fibrous article is the highest numbered test liquid for which two of the three drops remained spherical to

hemispherical, with no wicking for 30 seconds. In general, treated fibrous articles with a rating of 6 or more are considered good to excellent; fibrous articles having a rating of one or greater can be used in certain

applications.

Standard Test Liquids for Oil Repellency

Table 2

Fluorine measurement

Fluorine was analyzed as described by E. I. DuPont de Nemours and Co. Method No. Z4450.205.01 .CW as published on May 1 1 , 2005, also known as wichbold torch.

Suppliers

Capstone® TC was from (E. I. du Pont de Nemours and Company, Wilmington, Delaware, United States).

Urethane used in Examples herein has been disclosed in Examples 1 -23 in the US Application Serial No 14/226892, which is hereby incorporated as if fully set out herein.

Milli-Q water was obtained from EMD Millipore (Billerica, Massachusetts, United States) Despatch Protocol Plus™ convection oven (Minneapolis, Minnesota, United States)

Richard 80101 1 " foam brush was from Tools A. Richard Co.,

(Berthierville, Quebec, Canada)

Bovine serum albumin (BSA) was from Sigma Aldrich (St. Louis, Missouri, United States)

Fabrics used were 100% by weight khaki cotton twill from SDL Atlas Textile Testing Solutions, Rock Hill, South Carolina, U.S.

SDS surfactant is from Sigma-Aldrich, St. Louis, MO.

3 kDalton cut-off spin filters (Life Technologies, Grand Island, NY).

Bicinchoninic acid protein assay (BCA) kit is from Pierce/ThermoScientific, Rockford, IL.

Silver staining kit is from ThermoScientific, Rockford, IL.

EXAMPLE 1

TREATING FIBROUS ARTICLES WITH HYDROPHOBIN FOAM

This Example was designed to demonstrate foam treating using amphiphilic proteins (e.g., class II hydrophobin or Bovine serum albumin) a fluorinated polymer (e.g., Capstone® TC) or a non-fluorinated polymer (e.g., polyurethane).

Control sample

A 6 wt% Capstone® TC mixture was prepared by combining Milli-Q water (23.5 g) and Capstone® TC (1 .5 g) in a 50-ml plastic tripod beaker. The mixture was mixed thoroughly for several minutes with a metal spatula. Two samples of this mixture were prepared. Two cotton fibrous articles (4.5 x 3.5") were immersed into the solution individually for 1 minute. One sample (control 1 ) was hand-wrung, while the second sample (control 2) after hand-wringing, was blotted with a paper towel to remove the excess solution. The weight of the fibrous articles before and after dip and squeeze had been recorded. Each fibrous article sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven. Test sample with class II hydrophobin

A 0.01 wt% class II hydrophobin (HFBII) and 6 wt% Capstone® TC mixture was prepared by combining Milli-Q water (46.941 g), 8.5% ASP 20128085 HFBII hydrophobin (0.059g g, 61 .4 μΙ), and Capstone® TC (3 g) in a 250-ml plastic beaker. After gently swirling the mixture, a milk frother was used for 2 minutes to foam and the foam mixture was allowed to stand for 2 minutes to allow separation of the foam from the liquid. A sample of the foam and a sample of the liquid separated from the foam were analyzed as described above. Results obtained indicated that the foam contained 4410 ppm and the liquid contained 4506 ppm of fluorine.

Three separate samples were prepared as described above. In one sample, the foam was allowed to stand for 1 minute. In the second sample, the foam was allowed to stand for 4 minutes. In the third sample, the foam was allowed to stand for 7 minutes before they were applied onto three (3) separate cotton fibrous article pieces (4.5 x 3.5 "), using a Richard 80101 1 " foam brush. During application, each cotton fibrous article was placed on a dry paper towel to absorb excess moisture. Each foam was used to coat only one side of the fibrous article. The weight of the fibrous articles before and after foam application was recorded. Each fibrous article sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven.

Test sample with bovine serum albumin (BSA)

A 0.01 wt% BSA and 6 wt% Capstone® TC mixture was prepared by combining Milli-Q water (46.941 g), 8.5% ASP 20128085 HFBII hydrophobin (0.059g g, 61 .4 μΙ), and Capstone® TC (3 g) in a 250-ml plastic tripod beaker in the order listed. After gently swirling, the mixture was foamed for 2 minutes using a milk frother. The foam was allowed to stand for 1 , 4, and 7 minutes, in order to create a foam. The foam was then applied onto three (3) cotton fabrics (4.5 x 3.5 "), respectively, using a Richard 80101 1 " foam brush. During application, each cotton fabric was placed on a dry paper towel to absorb excess moisture, and only one side was coated. The weight of the fabric before and after foam application was recorded. Each fabric sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven. Test sample with both class II hvdrophobin and bovine serum albumin

A 0.01 wt% HFBII, 6 wt% Capstone® TC, and 0.005 wt% BSA mixture was prepared by combining Milli-Q water (46.939 g), 8.5% ASP 20128085 HFBII hydrophobin (0.059g g, 61 .4 μΙ), Capstone® TC (3 g), and BSA protein (0.0025 g) in a 250-ml plastic beaker in the order listed. After gently swirling, the mixture was foamed for 2 minutes using a milk frother. The foam was separated into 3 samples and each were allowed to stand for 1 , 4, and 7 minutes as described above. Each foam sample was then applied onto three (3) pieces of cotton fibrous article (4.5 x 3.5") using a Richard 80101 1 " foam brush. During application, each cotton fibrous article was placed on a dry paper towel to absorb excess moisture. Only one side of each fibrous article piece was coated with the foam. The weight of the fibrous articles before and after foam application was recorded. Each fibrous article sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven.

Production of class II hvdrophobin foam

A 0.1 wt% HFBII solution was prepared by combining Milli-Q water (24.706 g), and 8.5% ASP 20128085 HFBII hydrophobin (0.294 g g, 307 μΙ) in a 250-ml plastic tripod beaker in the order listed. After gently swirling, the mixture was foamed for 2 minutes using a milk frother. The foam was allowed to stand for 1 minute as described above before applying onto cotton a fabric (4.5 x 3.5 ") using a Richard 80101 1 " foam brush. During application, the cotton fabric was placed on a dry paper towel to absorb excess moisture, and only one side was coated. The weight of the fabric before and after foam application was recorded. The fabric sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven.

Polyurethane solution (control)

A 6 wt% polyurethane mixture (as a non-fluorinated textile effect additive) was prepared by combining Milli-Q water (23.5 g), and

polyurethane (1 .5 g) in a 50-ml plastic tripod beaker. The mixture was mixed thoroughly for several minutes with a metal spatula. A cotton textile (4.5 x 3.5") was immersed into the solution individually for 1 minute. The sample was hand-wrung to remove excess liquid. The weight of the fabric before and after dip and squeeze was recorded. The fabric sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven.

Hydrophobin II and polyurethane foam

A 0.01 wt% HFBII and 6 wt% polyurethane mixture was prepared by combining Milli-Q water (46.941 g), 8.5% ASP 20128085 HFBII hydrophobin (0.059 g, 61 .4 μΙ), and polyurethane (3 g) in a 250-mL plastic tripod beaker in the order listed. After gently swirling, the mixture was foamed for 2 minutes using a milk frother. The foam was allowed to stand for 1 minutes as described above before applying onto a cotton fabric (4.5 x 3.5 "), using a Richard 80101 1 " foam brush. During application, the cotton fabric was placed on a dry paper towel to absorb excess moisture, and only one side was coated. The weight of the fabric before and after foam application was recorded. The fabric sample was cured at 165°C for 5 minutes in a Despatch Protocol Plus™ convection oven.

Each fabric sample was then tested for its water repellency and oil repellency by methods described above. The results are shown in Table 3 below. Higher numbers indicated water and/or oil repellency. Table 3

6 wt% (7 min)

Capstone® TC

BSA 0.01 wt% BSA Foam treating 75% 7 5

6 wt% (1 min)

Capstone® TC

BSA 0.01 wt% BSA Foam treating 70% 7 5

6 wt% (4 min)

Capstone® TC

BSA 0.01 wt% BSA Foam treating 74% 7 5

6 wt% (7 min)

Capstone® TC

HFBII+BSA 0.01 wt% HFBII Foam treating 77% 7 6

0.005 wt% BSA (1 min)

6 wt%

Capstone® TC

HFBII+BSA 0.01 wt% HFBII Foam treating 70% 7 6

0.005 wt% BSA (4 min)

6 wt%

Capstone® TC

HFBII+BSA 0.01 wt% HFBII Foam treating 78% 7 6

0.005 wt% BSA (7 min)

6 wt%

Capstone® TC

HFBII only 0.1 wt% HFBII Foam treating 65.3 1 -2 1 foam (1 min)

polyurethane 6 wt% Dip and Squeeze 1 18 4 1 control polyurethane (gentle)

solution

polyurethane 0.01 wt% HFBII, Foam treating 76.6 4 1 + HFBII 6 wt% (1 min)

polyurethane

foam

The results shown in Table 3 indicate that while addition of the class II hydrophobin or BSA to the samples containing the fluorinated polymer, Capstone® TC or polyurethane textile effect additives, did not adversely affect water and oil repellencies of the fibrous articles, it reduced their wet pick up as evidenced by the reduced numbers for wet pick up by these fabrics. EXAMPLE 2

DETERMINATION OF THE FOAM DENSITY (AMOUNT OF WATER IN

THE FOAM)

The foam used in Example 1 was prepared by preparing a mixture of 0.01 wt% HFBII and 6 wt% Capstone TC or 0.01 wt% HFBII and 6 wt% polyurethane to give a final mass of 100 g (M0). The mixture was foamed for 3 minutes in a 300-ml glass beaker. After 3 minutes, the foam/water mixture was allowed to stand for 5 to seconds to facilitate the separation of water from foam. The volume of the foam was recorded (V1 ). The water was then carefully decanted from the beaker and weighed (M1 ) to determine the weight of the foam in the beaker (M0-M1 ), from which the density of the wet foam was determined using formula (M0-M1 )A/1 . The foam was then spread onto paper towel for 2 minutes to dry. After this time, the dry foam was carefully removed from the paper towel using wooden tongue depressor, and placed into a clean glass beaker. The weight was recorded (M2), and the volume was estimated (V2). The density of dry foam was calculated from the formula M2A/2. Table 4 below shows the foam density for hydrophobin II formulations.

Table 4

Foam density for HFBII formulations

Foam composition Dry foam Wet foam

0.01 wt% HFBII, 6 wt% 0.0317 g/ml 0.2190 g/ml

Capstone® TC

0.01 wt% HFBII, 6 wt% 0.0491 g/ml 0.1004 g/ml urethane

EXAMPLE 3

SEM CHARACTERIZATION OF TREATED COTTON FABRICS

The microstructures of foam treated cotton fabrics were

characterized using SEM. The SEM images of a blank cotton and three hydrophobin-Capstone® TC treated cottons are shown in Figures 1 A and 1 B. The first row of Figure 1A shows a blank cotton sample and subsequent rows show treated cottons with increasing Capstone® TC loadings. From left, middle to right columns are pictures with increasing magnifications (x30, x1000, x3000). Based on the low magnification pictures (x30), the treatments did not alter the texture or appearance of the fabric at macroscopic level. Under higher magnifications (x1000 and x3000), it appears that the cotton fibers in the treated fabrics becomes smoother with increasing Capstone® TC loadings, indicating the formation of thin, uniform coherent polymer coating on each individual fibers.

EXAMPLE 4 (PROPHETIC)

DETERMINATION OF THE CONCENTRATION OF CLASS II HYDROPHOBIN ON THE FIBROUS ARTICLE

To obtain an estimate for the concentration of the HFB residue on the foam coated fabric, HFB is removed from the fabric by repeated washes with 1 wt% SDS surfactant solution. The dissolved hydrophobin is collected and concentrated with 3 k Dalton cut-off spin filters. The hydrophobin concentration is then analyzed using the BCA protein assay which allows determination of the HFB concentration based on its absorption at 562 nm with reference to a set of hydrophobin standards.

Alternatively, the HFB removed from the fabric can be quantified using SDS-PAGE gel electrophoresis well known in the art. The hydrophobin sample and standards at various concentrations is mixed with appropriate amounts of loading dye and DTT. The mixtures are then loaded onto SDS-PAGE gel for electrophoresis. After the completion, the gel is stained using the Silver staining kit.