Title of Invention

"A NOVEL DYE COMPOSITION PREPARED FROM PLANT PARTS OF LAWSONIA INERMIS AND THE METHOD OF PREPARATION THEREOF"

Abstract

The present invention provides a dye composition prepared from Lawsonia inermis for staining proteins on a gel. The dye composition of the present invention comprises petrol based extract of henna leaves, ammonia, lemon juice and ethanol. The present invention also provides a method for preparing the dye composition and further provides a method for conducting sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay using the said dye which is advantageous over the conventionally followed methods.

Full Text

Field of the Invention: The present invention relates to a dye composition prepared from Lawsonia inermis for staining proteins on a gel. The present invention also provides a method for preparing the said dye composition. The present invention further provides a method for conducting sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay using the said dye. Background and Prior Art: There are several methods which have been developed in the past for separating proteins according to their size. One of the most commonly used methods for separating the proteins is the SDS-PAGE assay. The general set up of the apparatus for performing the SDS-PAGE assay is illustrated in Figure 1. The purpose of SDS-PAGE assay is to separate proteins according to their size, and no other physical feature. Sodium Dodecyl Sulfate (SDS) SDS is used to denature all proteins to the same linear shape. SDS is a detergent (soap) that can not only dissolve hydrophobic molecules but also has a negative charge (sulphate) attached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be soluablized by the detergent, plus all the proteins will be covered with many negative charges. This is known as SDS "wrapping around" the polypeptide backbone. SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. The effect of treating the sample solution containing protein is illustrated in Figure 2. Poly Acrylamide Gel Electrophoresis (PAGE) If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size. So we need to put the proteins into an environment that will allow different sized proteins to move at different rates. The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium. Adding a gel with sieving properties (that is a gel where the resistance to the motion of a particle increases with particle size) increases the differences in mobility between proteins of different molecular weights. This is the basis of separation. The environment of choice is polyacrylamide, which is a polymer of acrylamide monomers. The structure of the polyacrylamide gel is illustrated in figure 3. Thus, in the SDS-PAGE assay, the sample protein which is to be separated is first denatured using SDS and then loaded on the gel and to the gel electric field is applied thereby facilitating separation of the protein molecules. In order to facilitate viewing of the proteins thus separated, the gel is stained with a dye or a stain. The stain contained in the gel binds with the denatured protein and moves along with the same thereby facilitating viewing of the separation of the protein. Several procedures are now available for staining the proteins. Also, several different types of stains are available which can be used for staining the protein. Some of the commonly used stains include Coomassie Brilliant Blue R-250, Amido Black and Silver stain. United States Patent no. 6,057,160 discloses a rapid and simple method for making a hydrophobically associated, Coomassie Brilliant Blue (CBB) dye/protein complex comprising CBB and a protein, and a method for detecting analytes using the CBB dye/protein complex. Korean patents no. 445939 describes the use of SeePico™ CBB Stain Kit. The patent is pending in other countries (including U.S.A, Japan, E.U., China, Australia).The Kit makes use of the newly developed technique (patent pending) using Coomassie Brilliant Blue G-250 (CBB-G250) dye for protein staining in sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This innovative kit allows protein bands or spots to be viewed with hypersensitivity (1ng or less of protein bands can be detected) from 30 minutes. The Coomassie dye (Brilliant blue G250) whose structure is shown in figure 4, binds to protein molecules in acid pH by two means. The triphenylmethane group binds to nonpolar structures in proteins, and the anion sulfonate groups interact with protein cationic side chains (e.g., arginine and lysine side chains) in acid pH. The color change produced when the dye binds to proteins provides a measure of total protein, which is quite sensitive in the case of albumin and certain globular proteins. The Coomassie dye-binding assay, or Bradford assay, also responds to some interfering substances which are generally unknown unless specifically tested for. Nevertheless, because of its apparent simplicity and sensitivity towards many proteins, the Bradford assay is popular and widely used. The salient features of the Coomassie dye-binding assay are: • It develops intensely colored complexes with proteins • It can determine as little as 0.5 µg/cm2 of protein present in a gel matrix • Anion of Coomassie Brilliant Blue formed in the acidic staining medium combines with the protonated amino groups of proteins by electrostatic interaction; • resulting complex is reversible under the proper conditions Although the Coomassie dye-binding assay is widely used, it has got severe safety related disadvantages. The following is a quote from the Material safety data sheet of national diagnostics with regard to coomassie brilliant blue R-250: "IT MAY BE HARMFUL IF SWALLOWED OR INHALED. MAY CAUSE IRRITATION TO SKIN AND EYES. THE TOXICOLOGICAL PROPERTIES OF THIS SUBSTANCE HAVE NOT BEEN FULLY INVESTIGATED." Some of the potential health risks of using coomassie brilliant blue R-250 include: o Inhalation: Causes irritation to the respiratory tract. o Ingestion: No information found, but compound should be handled as a potential health hazard. May cause irritation to the digestive tract. o Skin: May cause irritation, redness and pain. o Eyes: May cause irritation, redness and pain. In addition to the above mentioned safety related disadvantages, some of the other disadvantages include: • the price of the dye is high; • it is not readily available at all places. It has to be procured from stores and cannot be prepared at home; • the time taken for de-staining the gel is very long. It takes about 15 hours to de-stain the gel. In view of the above disadvantages, it is required to provide an alternative dye or stain which is at least equivalent, if not better than the commonly available dyes including the Coomassie dye in terms of sensitivity and overcomes at least one of the disadvantages described above. Object of the Invention: The main object of the present invention is to provide a novel dye for staining proteins on a gel. Another object of the present invention is to provide a method for preparing the dye. Yet another object of the present invention is to provide a method for conducting sodium- dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay using the said dye. Statement of the Invention: A novel dye composition prepared from plant parts of Lawsonia inermis substantially as herein described with reference to the foregoing example and the accompanying drawings. However, the embodiments given are just illustrative in nature and do not limit the scope of the invention in terms of its application, or uses. Summary of the Invention: Accordingly, the present invention provides a dye composition prepared from Lawsonia inermis for staining proteins on a gel. The dye composition of the present invention comprises petrol based extract of henna leaves, ammonia, lemon juice and ethanol. The present invention also provides a method for preparing the dye composition and further provides a method for conducting sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay using the said dye which is advantageous over the conventionally followed methods. Brief Description of the Accompanying Drawings: In the drawings accompanying the specification, Figure 1 illustrates the general set up of the apparatus for performing the SDS-PAGE assay. Figure 2 illustrates the effect of treating the sample solution containing protein with SDS. Figure 3 illustrates the general structure of the polyacrylamide gel. Figure 4 illustrates the chemical structure of Coomassie dye (Brilliant blue G250). Figure 5 illustrates the chemical structure of lawsone. Figure 6 illustrates the manner of loading the sample in the well in accordance with experiment 13. Figure 7 illustrates the bands formed after conducting experiment 13. Figure 8 illustrates the manner of loading the sample in the well in accordance with experiment 14. Figure 9 illustrates the bands formed after conducting experiment 14. Figure 10 illustrates the manner of loading the sample in the well in accordance with experiment 15. Figure 11 illustrates the bands formed after conducting experiment 15. Figure 12 illustrates the manner of loading the sample in the well in accordance with experiment 16. Figure 13 illustrates the bands formed after conducting experiment 16. Figure 14 illustrates the manner of loading the sample in the well in accordance with experiment 17. Figure 15 illustrates the bands formed after conducting experiment 17. Figure 16 illustrates the manner of loading the sample in the well in accordance with experiment 18. Figure 17 illustrates the bands formed after conducting experiment 18. Figure 18 illustrates the manner of loading the sample in the well in accordance with experiment 19. Figure 19 illustrates the bands formed after conducting experiment 19. Figure 20 illustrates the manner of loading the sample in the well in accordance with experiment 20. Figure 21 illustrates the bands formed after conducting experiment 20. Figure 22 illustrates the manner of loading the sample in the well in accordance with experiment 21. Figure 23 illustrates the bands formed after conducting experiment 21. Figure 24 illustrates the manner of loading the sample in the well in accordance with experiment 22. Figure 25 illustrates the bands formed after conducting experiment 22. Figure 26 illustrates the manner of loading the sample in the well in accordance with experiment 23 and the bands formed after conducting the experiment. Figure 27 illustrates the manner of loading the sample in the well in accordance with experiment 24 and the bands formed after conducting the experiment. Figure 28 illustrates the manner of loading the sample in the well in accordance with experiment 31 and the bands formed after conducting the experiment. Figure 29 illustrates the manner of loading the sample in the well in accordance with experiment 32 and the bands formed after conducting the experiment. Detailed Description of the Invention: Accordingly, the present invention provides a dye composition prepared from plant parts of Lawsonia inermis, said dye composition comprising 15 to 25% v/v of petroleum based paste or extract of the plant parts of Lawsonia inermis, 35 to 45 % v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia and 17 to 23 % v/v of alcohol and optionally a source of sugar. In an embodiment of the present invention, the acid is selected from the group comprising lemon juice, galgal/hill lemon juice and lime juice. In another embodiment of the present invention, the acid is preferably lemon juice. In yet another embodiment of the present invention, the amount of lemon juice present is about 40 % v/v. In still another embodiment of the present invention, the source of ammonia is selected from the group comprising ammonium hydroxide, liquid ammonia and ammonium sulphate. In a further embodiment of the present invention, the source of ammonia is preferably liquid ammonia. In one more embodiment of the present invention, the amount of liquid ammonia present is about 20 % v/v. In an embodiment of the present invention, the alcohol is selected from the group comprising methanol, ethanol, propanol and butanol. In another embodiment of the present invention, the alcohol is preferably ethanol. In yet another embodiment of the present invention, the amount of ethanol present is about 20 % v/v. In still another embodiment of the present invention, the source of sugar is a selected from the group comprising glucose, sucrose, maltose and lactose. In one more embodiment of the present invention, the source of sugar is preferably sucrose. In a further embodiment of the present invention, the source of sugar is present in a quantity in the range of 12 to 20 % w/v and preferably the quantity of sugar present is 16 % w/v. In an embodiment of the present invention, the plant parts of Lawsonia inermis are selected from the group comprising leaves and tender stem. In another embodiment of the present invention, the petroleum based extract /paste is selected from the group comprising petrol based extract or petrol based paste. The present invention also provides a process for preparing the dye composition from plant parts of Lawsonia inermis, said process comprising the steps of: (a) extracting the plant parts of Lawsonia inermis using a petroleum solvent to obtain a petroleum based paste or a petroleum based extract; (b) adding to the petroleum based paste or the petroleum based extract thus obtained in step (a) 35 to 45% v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia, 17 to 23 % v/v of alcohol and optionally a source of sugar to obtain a mixture; and (c) allowing the mixture obtained in step (b) to stand for a time period in the range of 20 to 28 hours and decanting the same to obtain the dye composition. In an embodiment of the present invention, the plant parts of Lawsonia inermis are selected from the group comprising leaves and tender stem. In another embodiment of the present invention, the petroleum based extract /paste is selected from the group comprising petrol based extract or petrol based paste. In yet another embodiment of the present invention, wherein in step (a) about 120 gms of wet Lawsonia inermis leaves stored at -20°C is ground and about 20 ml of petrol is added to the same and thoroughly mixed to obtain petrol based paste. In still another embodiment of the present invention wherein in step (b), the acid is selected from the group comprising lemon juice, galgal/hill lemon juice and lime juice. In one more embodiment of the present invention wherein in step (b), the acid is preferably lemon juice. In a further embodiment of the present invention wherein in step (b), the amount of lemon juice added is preferably about 25 ml. In an embodiment of the present invention wherein in step (b), the source of ammonia is selected from the group comprising ammonium hydroxide, liquid ammonia and ammonium sulphate. In another embodiment of the present invention wherein in step (b), the source of ammonia is preferably liquid ammonia. In yet another embodiment of the present invention wherein in step (b), the amount of liquid ammonia added is preferably about 12.5 ml. In still another embodiment of the present invention wherein in step (b), the alcohol added is selected from the group comprising methanol, ethanol, propanol and butanol. In one more embodiment of the present invention wherein in step (b), the alcohol is preferably ethanol. In a further embodiment of the present invention wherein in step (b), the amount of ethanol added is preferably about 12.5 ml. In an embodiment of the present invention wherein in step (b), the source of sugar added is a selected from the group comprising glucose, sucrose, maltose and lactose. In another embodiment of the present invention wherein in step (b), the source of sugar added is preferably sucrose. In yet another embodiment of the present invention wherein in step (b), the quantity of sugar added is in the range of 12 to 20 % w/v and preferably the quantity of sugar added is preferably about 10 gms. The present invention further provides a method for conducting sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay, said method comprising the steps of: (a) preparing denatured protein sample; (b) preparing polyacrylamide gel; (c) loading the denatured protein sample on to the polyacrylamide gel and applying electric field across the same; (d) staining the polyacrylamide gel of step (c) using a dye composition comprising 15 to 25 % v/v of petroleum based paste or extract of the plant parts of Lawsonia inermis, 35 to 45 % v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia and 17 to 23 % v/v of alcohol and optionally a source of sugar to obtain stained polyacrylamide gel; and (e) de-staining the stained polyacrylamide gel. In an embodiment of the present invention wherein in step (c), the polyacrylamide gel is stained using a dye composition for a time period in the range of 30 to 35 minutes at temperature in the range of 40° to 45°C. In another embodiment of the present invention, the step of de-staining the stained polyacrylamide gel comprises: (i) treating the stained polyacrylamide gel with acetic acid having a first concentration level for a first time period to obtain a partially de-stained polyacrylamide gel; and (ii) treating the partially de-stained polyacrylamide gel thus obtained with acetic acid having a second concentration level for a second time period to obtain substantially de-stained polyacrylamide gel. In yet another embodiment of the present invention, the first concentration level of acetic acid is in the range of 45 to 55% and is preferably about 50%. In still another embodiment of the present invention, the value first time period is in the range of 20 to 35 min and preferably in the range of 25 to 30 minutes. In one more embodiment of the present invention, the second concentration level of acetic acid is in the range of 5 to 10 % and is preferably about 7%. In a further embodiment of the present invention, the value second time period is in the range of 70 to 100 min and preferably in the range of 80 to 90 minutes. In a further more embodiment of the present invention, step (ii) is preferably performed at elevated temperature and more preferably performed at temperature in the range of 65° to 70°C. Detailed Description of the Invention Lawsonia inermis commonly known as Henna, grows in clusters of small, intensely fragrant flowers. Sometimes the flowers are pink or red, but usually they are white. Three species of the Lawsonia plant are used as henna: Inermis, Alba and Spinoza. Belongs to family lythraceae Modern scientists have many beneficial properties in Henna. In fact, the actual Henna plant is sometimes called the "Magic Plant" because it has great healing effects. It contains ingredients to be antibacterial, anti-fungal, and anti-hemorrhagic. Henna is useful is healing athlete's foot, fungal skin infections, headaches, burning of the soles and palms, and local inflammation. The leaves and seeds of the plant possess medicinal properties. They both act as cooling agents for the head and body. Henna will only grow where the minimum temperature is above 50° F or 11° C. Henna is a small tree and has to grow for 5 years to mature and produce leaves with useful levels of tannin. Therefore, for henna to grow outdoors unsheltered, the temperature must stay above 50° F for many years. Henna grows better in arid regions than wet regions. Henna produces higher levels of tannins where the maximum temperature is very high. Henna produces higher levels of tannins in very arid climates than in moist climates. The hottest, driest regions have henna with the highest levels of tannin that make the darkest stains. Henna from these areas can be dried, powdered, and sold. If henna is from a moist region, the henna is good for use fresh, but is not good for use dried and powdered, because of the lower tannin levels. Constituents of henna  Hennatonic acid (A Natural protein)  Lawsone (A natural pigment)  Mannitol  Volatile Oil  Fats  Resin Staining action of henna on stratum corneum Skin is our body's largest organ. It keeps our body's moisture inside our body so we don't die of dehydration. The stratum corneum is the outermost membrane of our skin. Our skin also keeps injurious exogenous agents outside the body. The stratum corneum is made of keratin, the same protein that makes hair, fingernails, horn, hooves and turtle shells. On some parts of our body, the stratum corneum is very thick, and on other parts it is thin. The thickest skin is on our heels, where the stratum corneum averages 86 layers of cells. Every day our body grows new layer of skin cells below the surface of the skin. These skin cells are interlocked and rise upward in columns, being pushed up by the new cells underneath. As they rise in this column, they gradually die, dry out and become "corneated". Thus, instead of being moist, soft and flexible, they get stiff and dry. When they emerge at the surface of skin, they are very dead, very dry, and getting loose. Dry and flaky skin is an indication of heavy shedding of corneated cells from Stratum Corneum. Henna has a tannin dye molecule, lawsone, which is a natural pigment or in other words the dye molecules. It is believed that lawsone is small enough to penetrate a skin cell and imparts the color. If we put henna paste on skin, the dye molecules will penetrate down the columns of skin cells. They don't spread out, as ink would on blotter paper. On the other hand, they go straight down as ink would on corrugated cardboard. That is why the pattern stays clear and in place till the last day of exfoliation. When the henna pattern disappears, the henna stained cells have emerged at the outside of the skin and exfoliated. Tattooed cells are deeper in the skin, and do not exfoliate. To tattoo skin, pigment is placed under the Stratum Corneum into the basal and spinous layers. That is why tattoos bleed and hurt. Tattoo pigment is going into living tissue. Henna does not hurt because it is staining dead cells, and those cells exfoliate. Henna releases its dye content in acidic conditions. That means we can get the dye content by providing some acid to the pure henna. Commonly used acid is citric acid, i.e., is lemon. Henna releases its dye content at a pH of 5.5. On oxidation the stain gets darker. Air oxidizes the henna stain. We can also force oxidation by putting something alkaline on the surface of skin. Sweat is a safe alkaline. In place of sweat, limestone can also be used to provide alkaline conditions. Henna stains on skin and hair darken during the first 48 hours after paste removal. If keratin is saturated with lawsone, under certain conditions the darkening can be dramatic. If silk is stained with henna, and the cloth is heated by ironing, the stain will become dark brown. If palm or sole skin is saturated with henna, steam will darken the henna to dark brown or near black. If a henna-stained palm is treated with dilute ammonia or another alkaline, the stain will darken to brown-black or greenish black. Chemically, the molecule of lawsone, whose structure is illustrated in figure 5, is 2-hydroxy-l,4-naphthoquinone. Industrial classifications also describe lawsone as Natural Orange 6 and C.I. 75480. Lawsone show its congeniality to naphthalene. In lawsone, two oxygen atoms are attached to the naphthalene carbons at positions 1 and 4 to form 1,4-naphoquinone and a hydroxyl group is present at position 2. Its molecule contains 10 carbons, 6 hydrogens and 3 oxygens (C10H6O3), giving a total molecular weight of 174.16 atomic units of mass. Pure lawsone is an orange powder, insoluble in water, with a melting point higher than 192°C and optical absorption maximum of 452 nm. As the structure of lawsome is not similar to the structure of Coomassie dye, there exists no reason for a person skilled in the art to presume that lawsome can be used as a replacement of Coomassie dye. Thus, the complete tests of finding a substitute for Coomassie dye were conducted without any clue from any literature guidance. The details of the tests conducted are outlined here below. It should however, be understood that the following paragraphs are provided merely to enable a person skilled in the art to better understand the invention and nothing contained in the following sections is intended to limit the scope of the invention in any manner. The scope of the claims is intended to be limited by only the claims and their equivalent. For the conducting the SGS-PAGE assay, the following chemicals are required:  Acrylamide (30%)  Lower gel buffer  Upper gel buffer  Running buffer  TEMED  APS  SDS Preparation of Different Types of Solutions: (a) Preparation of Stock acrylamide (30%): To about 73 g acrylamide 1.95 g Bis acrylamide is added and the mixture thus obtained 250 ml of distilled H2O is added. (b) Preparation of Sample buffer: To 0.72 g Tris base add 6.25 g glycerol (pH to 6.8), 2.5 mg bromophenol blue and 2.5 g SDS. To the mixture thus obtained add distilled H2O. The amount of the distilled H2O added would depend upon the requirement. By way of example, add 25ml of distilled H2O for obtaining a concentration of about 5X and add 125ml of distilled H2O for obtaining a concentration of about 1X. (c) Preparation of Running buffer stock: To about 3 x 202 g Tris-OH add 10 x 288 g Glycine and 2 x 100 g SDS. To the mixture thus obtained add about 20 liter of distilled water on carboy. The pH need not be adjusted as the pH can be between 8.3 to 8.7. In order to check the pH, preferably use pH paper instead of using pH meter. d) Preparation of Lower gel buffer, LGB: To about 45.43 g Tris base add 5.75 ml concentrated HC1. If necessary, adjust the pH to 8.8. Add to the mixture thus obtained 1 g SDS and about 250 ml of distilled water. (e) Preparation of Upper gel buffer, UGB: To 7.58 g Tris base add about 4.8 ml of concentrated HC1. If necessary, adjust the pH value of the mixture to about 6.8. Add about 0.5 g SDS and 125 ml of distilled water. (f) Preparation of Ammonium persulfate: Add about 100 mg of Ammonium persulfate per ml distilled H2O. (g) Preparation of Coomassie blue stain and fixative: To about 500 ml of methanol add 400 ml distilled H2O and 100 ml glacial acetic acid. To the above mixture add 1 g Coomassie Brilliant Blue R-250. The stain may be reused multiple times if the staining is too light or alternatively it can be discarded. In an alternate method which does not require de-staining, add about 5 ml of stain to about 300 ml of destain and place gel overnight and allow it to get dried for use the next morning. (h) Preparation of Destainer: To about 300 ml of methanol add 2.49 liter of distilled H2O and 210 ml glacial acetic acid. A small piece of foam sponge added speeds up de-staining and conserves de-stain solution. Gel preparation data Two glass plates, washed with ethanol (one notched) were placed on each other, separated by two separators known as spacers. Plates were joined by tape. Leakage was checked by pouring water in between two plates. Before pouring lower gel buffer, plug gel was prepared. Decanted water and poured plug gel .After polymerization of plug gel, lower gel (also called as separating gel) was poured. For the various experiments performed, 10% lower gel was used. Here we waited for the polymerization of the gel. pH of the lower gel is about 8.8. After polymerization of lower gel, upper gel (also called as stacking gel) was added. The pH of stacking gel is 6.8. After pouring upper gel; a comb is placed for the formation of wells. After polymerization, comb was removed and wells were rinsed with water. Remove the tape from bottom side so as to make the circuit complete. The gel is now ready for being placed on the plate on electrophorosis apparatus containing running buffer in upper and lower reservoirs. The compositions of the plug-in gel, the lower gel and the upper gel are provided in table 1, table 2 and table 3 respectively. Table 1: Composition of Plug-in Gel Table 2: Composition of Lower Gel Table 3: Composition of upper Gel (Table Removed) Performing the SDS-PAGE Assay: Once the gel is ready, preheated sample buffer containing samples and selectable marker, whose molecular weight was known is loaded in the well. After this electric field was applied. Proteins having negative charge move towards positive electrode at 100V and bands were formed. These bands were visible after staining and destaining of the gel with different dyes. The velocity of a charged particle moving in an electric field is directly proportional to the field strength and the charge on the molecule and is inversely proportional to the size of the molecule and the viscosity of the medium. Adding a gel with sieving properties (that is a gel where the resistance to the motion of a particle increases with particle size) increases the differences in mobility between proteins of different molecular weights. This is the basis of separation. A number of experiments were performed to evaluate different types of dye compositions containing henna for their suitability for the SDS-PAGE assay. Details of some of the types of the types of dye compositions containing henna leaves which were prepared, the method for preparing the dye compositions and the tests conducted to determine their suitability for the SDS-PAGE assay are outlined in the following example. Wherever possible, the Applicants have tried to provide comparative data with reference to Coomassie Brilliant Blue R-250 dye. The following examples are provided only by way of illustration and nothing contained in the following section should be construed to limit the scope of the present invention in any manner. The scope of the claims is intended to be limited merely by the scope of the claims in themselves and their equivalents. Experiment 1: Preparation of Dye 1 comprising Henna - Dried leaves 75g of dried henna leaves were crushed to make powder. Powder was mixed with 25 ml of double distilled water and 25 ml of ethanol. Mixture was homogenized and was kept at room temperature for one hour. Then it was filtered to obtain Dye 1. Experiment 2: Preparation of Dye 2 comprising Henna - Ca(OH)2- ethanol 150g of green henna leaves (stored at -20°C) were grinded in 20ml of 5% calcium hydroxide. After that 25ml of ethanol and same amount of water was added to the paste. It was then homogenized using a homogenizer. Homogenized paste was kept for 24 hours at room temperature. Thereafter, the same was filtered to obtain Dye 2. Experiment 3: Preparation of Dye 3 comprising Henna - Wet leaves (Stored at -20°C) Green henna leaves (stored at -20°C) were grinded. 75g of paste was further grinded with 25ml of ethanol and 25 ml water. The paste was then homogenized along with 25ml ethanol and 75ml water. After that 50 ml ethanol was added and it was left for 5 hours and filtered to obtain Dye 3. Experiment 4: Preparation of Dye 4 comprising Henna - Mixed leaves dye Green henna leaves (stored at -20°C) were grinded. To 75g of the paste thus obtained, 50g of dried henna powder were added to make it thicker and was further grinded with 25ml of ethanol and 25 ml water. The paste was then homogenized along with 25ml ethanol and 75ml water. After that 50 ml ethanol was added and it was left for 5 hours and filtered to obtain Dye 4. Experiment 5: Preparation of Dye 5 comprising Henna - Ammonia, Ethanol, Petrol and Lemon juice (leaves stored at -20°C) 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 25 ml of lemon juice and 12.5 ml each of ammonia and ethanol was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 5. Experiment 6: Preparation of Dye 6 comprising Henna -Normal dye (leaves stored at -20°C) 50 g of wet henna leaves were grinded and a paste was formed. To the paste 75 ml of ethanol and 25 ml of double distilled water was added. The constituents were further grinded and paste was heated at 40°C - 45°C for 15 - 20 minutes and kept at room temperature for 24 hours and filtered to obtain Dye 6. Experiment 7: Preparation of Dye 7 i.e. Dye 5 without petrol 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of water. To the paste formed 25 ml of lemon juice and 12.5 ml each of ammonia and ethanol was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 7. Experiment 8: Preparation of Dye 8 i.e. Dye 5 without ammonia 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 25 ml of lemon juice and 12.5 ml each of water and ethanol was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 8. Experiment 9: Preparation of Dye 9 i.e. Dye 5 without lemon juice 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 25 ml of water and 12.5 ml each of ammonia and ethanol was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 9. Experiment 10: Preparation of Dye 10 i.e. Dye 5 without ethanol 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 25 ml of lemon juice and 12.5 ml each of ammonia and water was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 10. Experiment 11: Preparation of Dye 11 i.e. Dye 5 without sugar 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 25 ml of lemon juice and 12.5 ml each of ammonia and ethanol was added. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 11. Experiment 12: Preparation of Dye 12 i.e. Dye 5 without lemon and ethanol 120 g of wet henna leaves (stored at -20°C) were crushed in 20 ml of petrol. To the paste formed 12.5 ml of ammonia was added. 10 g of sugar was also added to the paste. The constituents were mixed thoroughly and the paste was made to stand at room temperature for 24 hours and filtered to obtain Dye 12. Experiment 13 Gel Plate Preparation: Gel plates were prepared from the standard procedure as mentioned above. After the preparation of the gel plate the following procedure was followed. Sample preparation: Protein samples used for this experiment were BSA (Bovine serum albumin), chicken liver and egg. o BSA: 0.01g in 0.1ml of double distilled water. 100µl sample was added to 40µl sample buffer. o Chicken liver: 100µl liver solution was added to 40µl of sample buffer. o Egg: 100µl solution was added to 40µl of sample buffer. Loading: All the samples were heated and 40µl sample was loaded in each well of the plate as shown in figure 6 and experiment was started. Staining: Half of the gel was put in Dye 3 and half in Coomassie blue. Coomassie blue staining was done for half an hour and henna staining for 1 hour. De-staining: Gel stained with Dye 3 was put into the destainer (7% acetic acid) at 60-65°C in oven for one and a half hours. Overnight destaining of coomassie blue stained gel was done. Results: As shown in figure 7, bands were present in the gel stained with Dye 3. Liver protein was not visible both with coomassie and henna dye. Position of bands was same in both henna and coomassie stained gel. Experiment 14: To see the effect of using 7.5% gel and use the molecular weight markers while using the henna dye. Procedure: Gel plates were prepared from the standard procedure as mentioned previously. The concentration of lower gel was kept at 7.5%. After the preparation of the gel plate following procedure was followed. Sample preparation: Protein samples used for this experiment were molecular weight markers, BSA (Bovine serum albumin), chicken liver and egg. o BSA: 100mg/lml of double distilled water. 80µl sample was added to 40ul sample buffer. o Marker:10 µl of molecular weight markers were added to 4µl of sample buffer. o Chicken liver: 80µl liver solution was added to 40µl of sample buffer. o Egg: 80ui solution was added to 40µl of sample buffer. Loading: All the samples were heated and 30µl from each (molecular weight markers 12µl) was loaded in each well of the plate as shown in figure 8 and experiment was started. Staining: Half of the gel was put in Dye 3 and half in Coomassie blue. Coomassie blue staining was done for half an hour and henna staining for 1 hour. De-staining: Gel stained with Dye 3 was put into the destainer (7% acetic acid) at 60-65°C in oven for one and a half hours. Overnight destaining of coomassie blue stained gel was done. Results: As shown in figure 9, marker did not show the Dye 3 stain. The bands were lower as we have used 7.5% gel. Rest of the bands were same as that in coomassie blue. Experiment 15: To see the difference between the henna dye with and without calcium hydroxide. Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. After the preparation of the gel plate following procedure was followed. Sample preparation: Protein sample used for this experiment was BSA (Bovine serum albumin). Different concentrations of BSA used were: o 50mg/lml of double distilled water. o 100mg/lml of double distilled water. o 200mg/lml of double distilled water. o 500mg/lml of double distilled water. 80µl of each solution was mixed with 40µl of sample buffer. Loading: All the samples were heated and 30µl was loaded in each well of the plate as shown in figure 10 and experiment was started. Staining: Half of the gel was put in henna dye without calcium hydroxide and rest in henna dye made by suing calcium hydroxide. Both were stained for 1 hour. Destaining: Both gels were put into the destainer (7% acetic acid) at 60-65 °C in oven for one and a half hours. Results: As shown in figure 11 (a), Stain given by henna dye containing calcium hydroxide was not strong. Bands show white color when seen against black background. On the other hand, when henna dye that does not contain calcium hydroxide is used, the bands were brown in color as illustrated in figure 11 (b). Experiment 16: To see the effect of henna dye made with calcium hydroxide on wide range of protein samples. Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. After the preparation of the gel plate following procedure was followed. Sample preparation: Protein sample used for this experiment was BSA (Bovine serum albumin). Different concentrations of BSA used were: oBSA: 200mg/ml of double distilled water. 40µl sample was added to 20µl sample buffer. oBSA: 500mg/ml of double distilled water. 40µl sample was added to 20µl sample buffer o Chicken liver: 40µl liver solution was added to 20µl of sample buffer. o Egg: 40µl solution was added to 20µl of sample buffer. o Egg white: 40µl solution was added to 20µl of sample buffer. o Caesin: 10mg/ml of double distilled water. Loading: All the samples were heated and 20µl sample was loaded in each well as shown in figure 12. Staining: Gel was put in the henna dye made from calcium hydroxide. Bands were white in color after destaining. Destaining: Gel stained with henna was put into the destainer (7% acetic acid) at 60-65°C in oven for one and a half hours. Results: As sown in figure 13, bands representing the liver sample were not visible. Rest of the bands were visible. Experiment 17: To see the effect of destainer on the protein samples. Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was BSA (Bovine serum albumin). Different concentrations of BSA used were: o 50mg/lml of double distilled water. o 100mg/lml of double distilled water. o 200mg/lml of double distilled water. o 500mg/lml of double distilled water. 80µl of each solution was mixed with 40µl of sample buffer. Loading: All the samples were heated and 30µl was loaded in each well of the plate as shown in figure 14 and experiment was started. Staining: Half of the gel was put in Coomassie destainer and rest in henna destainer. Results: White bands were visible in both the cases within 15 minutes. This was due to the fixation of proteins and this also shows that the dye with Calcium hydroxide does not give stronger stain. Figure 15 (a) shows the results obtained when Coomassie de-stainer is used and figure 15 (b) shows the results when henna de-stainer is used. Experiment 18: To see the effect of different pH while using dye with calcium hydroxide. Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was BSA (Bovine serum albumin). Different concentrations of BSA used were: o 50mg/lml of double distilled water. o 100mg/lml of double distilled water. o 200mg/lml of double distilled water. o 500mg/lml of double distilled water. 80ul of each solution was mixed with 40µl of sample buffer. Dye: The pH was set at 2 and 5. Darkness of the dye color increases from 2 to 5. Loading: All the samples were heated and 30µl was loaded in each well of the plate as shown in figure 16. Three plates were made and were put in dye with different pH. Staining: Gels were put in dyes with different pH as shown in the figure. Results: Best results were given by the dye with pH - 5. In case of dye with pH - 5 bands were visible only after two and a half hours and same with dye at pH - 2. Bands of dye with pH - 2 as shown in figure 17 (a) were also good but lighter in color then pH - 5, which is shown in figure 17 (b). Experiment 19: To see the effect of different pH values on dye without calcium hydroxide. Procedure: Gel plates were prepared from the standard procedure as mentioned in chapter two. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was BSA (Bovine serum albumin). Different concentrations of BSA used were: o 10mg/ml of double distilled water. o 50mg/lml of double distilled water. o 100mg/lml of double distilled water. o 200mg/lml of double distilled water. o 500mg/lml of double distilled water. 80µl of each solution was mixed with 40µl of sample buffer. Dye: pH was set at 2, 5 and 11. Darkness of the dye color increases from 2 to 11. Loading: All the samples were heated and 30µl was loaded in each well of the plate as shown in figure 18. Different conditions of pH were used. Staining: Gels were put in dyes with different pH as shown in the figure. Results: As shown figure 19 (a) best results were given by the dye with pH - 11 and followed by pH - 5 which is shown in figure 19 (c). Dye with pH - 11 also took the least time to get destain, bands were visible in one and a half hours. Dye with pH -2 shown in figure 19 (b) did not give good bands. Experiment 20: To see the use of Coomassie blue and henna as dyes, compare the results and sensitivity by using serum sample. Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µl serum in 15 µl sample buffer. o 3 µl serum in 15 µl sample buffer. o 1 µl serum in 15 µl sample buffer. o 0.7 µl serum in 15 µl sample buffer. o 0.5 µl serum in 15 µl sample buffer. o 4 (ol of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µ1 of 20 times diluted serum sample in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figure 20 (a) and 20 (b). Staining: Gels were put in dyes as shown in the figure. Staining time for henna is 45 min - 60 min and for coomassie is 30 min. Destaining: Henna stained gel was destained for one and a half to hours at 65°C in 7% acetic acid. Coomassie stained gel was destained for overnight in its destainer. Results: As can be seen from figure 21 (a), Henna dye has shown the sensitivity up to 2.5 ug. The bands obtained by using coomassie blue is shown figure 21 (b). Experiment 21: To see the effect of ammonia on henna stain. Procedure: Gel plates were prepared from the standard procedure as mentioned in chapter two. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µl serum in 15 µl sample buffer. o 3 µl serum in 15 µl sample buffer. o 1 µl serum in 15 µl sample buffer. o 0.7 µl serum in 15 µl sample buffer. o 0.5 µl serum in 15 µl sample buffer. o 4 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µl of 20 times diluted serum sample in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figure 22. Staining: Gel was put in dye as shown in the figure. Staining time for henna is 45 min -60 min. Destaining: Henna stained gel was destained for one and a half to hours at 65°C in 7% acetic acid. Results: while figure 23 (a) represents serum samples stained in henna dye before applying ammonia, figure 23 (b) represents serum samples stained in henna dye after applying ammonia. For this purpose, after destaining the gel plate was put in ammonia for 5 minutes. As it can be noticed, when the destained gel is put in ammonia, the stain becomes darker. Therefore, it can be concluded that ammonia darkens the henna stain. Experiment 22: Compare the sensitivity of henna and coomassie blue dye along with the use of protein molecular weight markers. Procedure: Gel plates were prepared from the standard procedure as mentioned in chapter two. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µl serum in 15 µl sample buffer. o 3 µl serum in 15 µl sample buffer. o 1 µl serum in 15 µl sample buffer. o 0.5 |o.l serum in 15µl sample buffer. o 4 µ1 of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 15 µ1 of Protein molecular weight markers in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figures 24 (a) and 24 (b). Staining: Gel was put in dye as shown in the figure. Staining time for henna dye is 45 -50 minutes and for coomassie dye is 15-20 minutes. Henna stained gel was put in ammonia for 5 minutes before and after de-staining. Destaining: Henna stained gel was put in 7% acetic acid at 60°C - 65°C for one and a half to two hours. Coomassie stained gel was put in its destainer for overnight. Results: Figure 25 (a) shows the serum samples along with molecular weight stain marker stained in henna dye while figure 25 (b) shows the serum samples along with molecular weight stain marker stained in coomassie blue dye. Bands with 2.5 ug protein sample are visible both in henna and alta stained gels. But best results were with coomassie dye. One band of marker is visible in case of henna dye. In alta visibility of markers is better with best results in coomassie. Although results with coomassie dye seem better but destaining time taken in case of henna was very less. At higher protein content quality of bands with henna dye is better. Use of ammonia darkens the henna color. Experiment 23: Comparison of Dye 6 with Dye 5 Procedure: Gel plates were prepared from the standard procedure as mentioned above. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µl serum in 15 µl sample buffer. o 3 µl serum in 15 µl sample buffer. o 1 µl serum in 15 µl sample buffer. o 0.5 ul serum in 15 µl sample buffer. o 4 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 15 µl of Protein molecular weight markers in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figure 26. Staining: Two different gels were dyed, a first gel using Dye 6 and a second gel with Dye 5. Staining time for Dye 6 was 45 - 50 minutes at 40°C - 45°C and for Dye 5 was 45 to 50 minutes at 40°C - 45°C. Destaining: The gel stained with Dye 6 was put in 7% acetic acid at 60°C - 65°C for one and a half to two hours. The gel stained with Dye 5 was also destained for the same time under the identical conditions. Results: As shown in figure 26 (b), for Dye 6 the normal dye bands were good and 2.5 ug of protein sample was also visible after two hours of staining. In case of Dye 5 the visibility of bands was poor. It was not able to discriminate between the band and the other place. The complete gel was dark in color. However, when the gel dyed with Dye 5 was further de-stained, for three to four hours, the bands were visible. Color of bands with Dye 6 was yellowish brown while with Dye 5 was blackish brown. It can therefore be concluded that the amount of time period required for staining the gel should be lesser. If the gel is stained with Dye 5 for shorter period of time period, it would not only be possible to visualize the bands but also, the bands thus visualized would be substantially better in quality than what would be attained when Dye 6 is used. Experiment 24: Comparison of Dye 5 with Dye 6 with different staining and destaining conditions. Procedure: Gel plates were prepared from the standard procedure as mentioned previously. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µ1 serum in 15 µl sample buffer. o 3µl serum in 15 µl sample buffer. o 1 ul serum in 15 sample buffer. o 0.5µl serum in 15 µ1 sample buffer. o 4 µ1 of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 15 µl of Protein molecular weight markers in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figure 27 (a). Staining: Gel was put in Dye 5 and Dye 6. Staining time for Dye 6 was 45 - 50 minutes at 40°C - 45°C, while the staining time for Dye 5 was maintained between 30 - 35 minutes at 40°C - 45°C. Destaining: The gel stained with Dye 6 was put in 7% acetic acid solution at 60°C -65°C for one and a half to two hours. The gel stained with Dye 5 was destained with 50% acetic acid for 25 - 30 minutes and then with 7% acetic acid for 80 to 90 minutes at 65°C - 70°C temperature. Results: Under the above mentioned conditions, of staining and de-staining, better bands were formed with Dye 5. As shown in figure 27 (b), when Dye 5 is used, the contrast was good. In addition, Dye 5 also stained 2.5 µg of protein sample. Color of bands with Dye 5 was blackish brown while that with Dye 6 was yellowish brown (as shown in figure 27 (c)). Experiment 25: Staining and De-staining using Dye 7 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 7 was used for staining. Result-bands obtained were not clear and it was not able to stain protein having concentration of 0.1 and 0.25 µg. Experiment 26: Staining and De-staining using Dye 8 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 8 was used for staining. Result-Bands formed were very light. This dye was also not able to stain protein having concentration of 0.1 and 0.25 µg. Experiment 27: Staining and De-staining using Dye 9 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 9 was used for staining. Result-.This dye was not able to stain protein having concentration of 0.1 and 0.25 µg. Experiment 28: Staining and De-staining using Dye 10 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 10 was used for staining. Result-Bands formed were good but dye was not equally sensitive as that of Dye 5 or Dye 6. This dye was also not able to stain protein having concentration of 0.1 and 0.25 Experiment 29: Staining and De-staining using Dye 11 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 11 was used for staining. Result- Bands formed were equally good as that of Dye 5. Experiment 30: Staining and De-staining using Dye 12 Procedure: The various procedures such as Gel Preparation, sample preparation, loading procedure was kept identical to the procedure described in Experiment 24. The staining and the de-staining procedure adopted for Dye 5 in Experiment 24 was adopted with the difference that in place of Dye 5, Dye 12 was used for staining. Result- Bands formed were good but dye was not equally sensitive as that of Dye 5. This dye was also not able to stain protein having concentration of 0.1 and 0.25 ug. Conclusion: From experiments 25 to 30 performed, it can be concluded that all the constituents are important to stain the small amount of proteins like 0.1 ug. However sugar can be removed from the standard dye as results were equally good and dye 11 was equally sensitive as Dye 5 even after removing sugar. Experiment 31: : To use Dye 6, Dye 5 and Coomassie blue as dyes on protein serum samples in range of 360 µg - 2.5 µg and markers Procedure: Gel plates were prepared from the standard procedure as mentioned earlier. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 7 µl serum in 15 µl sample buffer. o 3 µl serum in 15 µ1 sample buffer. o 1 µl serum in 15µl sample buffer. o 0.5 ul serum in 15 µl sample buffer. o 4 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 1 µl of 20 times diluted serum sample in 15 µl of sample buffer. o 20 µl of Protein molecular weight markers in 15 µ1 of sample buffer. All the samples were heated at 60°C-70°C. Dye: Dye 5, Dye 6 and coomassie blue dye were taken for testing. Loading: Samples were loaded in each well of the three gel plates as shown in figure 28 (a). Staining: Each of the three different gels were dyed, a first gel using Dye 6 and a second gel with Dye 5 and a third one with coomassie blue dye. Staining time for Dye 6 was 50-55 minutes at 40°C - 45°C. Staining time for Dye 5 was 25 to 30 minutes at 40°C - 45°C while the staining time for coomassie blue dye was maintained as 30 to 35 minutes. Destaining: Gel stained with Dye 6 was de-stained by placing the same in 7% acetic acid at 60°C -65°C for one and a half to two hours. Gel stained with Dye 5 was first de-stained using 50% acetic acid for 35 to 40 minutes and then with 7% acetic acid for around one hour fifteen minutes at 60°C - 65 °C temperature. Coomassie stained gel was put in its destainer overnight. Results: As can be seen from figures 28 (b) to (d), 2.5 ug of protein sample was stained by all the dyes. Markers were visible in all the dyes. Figure 28 (b) indicates the picture of the gel which is stained using Dye 5, figure 28 (c) illustrates the picture of the gel stained using Dye 6 and figure 28 (d) illustrates the picture of the gel stained with coomassie blue dye. Experiment 32: To compare the ability of Henna dye (Dye 5)with Coomassie Brilliant Blue R- 250 w.r.t staining and destaining the gel having specific protein concentrations in the range of 51.5 µg - 0.1 µg Procedure: Gel plates were prepared from the standard procedure as mentioned previously. The concentration of lower gel was kept at 10%. Sample preparation: Protein sample used for this experiment was serum. o 1 µl serum in 15 µl sample buffer. o 0.5 µl serum in 15 µl sample buffer. o 50 µl of 200 times diluted serum sample in 15 µl sample buffer. o 20 µl of 200 times diluted serum sample in 15 µl sample buffer. o 10 µl of 200 times diluted serum sample in 15 µ1 of sample buffer. o 1 µ1 of 200 times diluted serum sample in 15 µl of sample buffer. o 0.5 µl of 200 times diluted serum sample in 15 µl of sample buffer. All the samples were heated at 60°C-70°C. Loading: Samples were loaded in each well of the plate as shown in figure 29 (a). Staining: Gel was put in Dye 5. Staining time was 30 - 35 minutes at 40°C - 45°C while the staining time for Coomassie blue dye was maintained as 30 to 35 minutes. Destaining: The gel was de-stained with 50% acetic acid for 25 - 30 minutes and then with 7% acetic acid for 80 to 90 minutes at 65 °C - 70°C temperature. Coomassie stained gel was put in its destainer overnight. Conclusion: As can be seen from figure 29 (b) and 29 (c), the samples of upto 0.1 ug of protein sample is clearly visible. 0.1 µg of protein sample was visible in both dyes i.e.Henna dye (Dye 5) and Coomassie Brilliant Blue R- 250 dye respectively. The foregoing detailed description has described only a few of the many possible implementations of the present invention. Thus, the detailed description is given only by way of illustration and nothing contained in this section should be construed to limit the scope of the invention. The claims are limited by the claims in themselves, including the equivalents thereof. WE CLAIM: 1. A Novel dye composition prepared from plant parts of Lawsonia inermis, said novel dye composition comprising 15 to 25% v/v of petroleum based paste or extract of the plant parts of Lawsonia inermis, 35 to 45 % v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia and 17 to 23 % v/v of alcohol and optionally a source of sugar. 2. The Novel dye composition as claimed in claim 1, wherein the acid is selected from the group comprising lemon juice, galgal/hill lemon juice and lime juice. 3. The Novel dye composition as claimed in claim 2, wherein the acid is preferably lemon juice. 4. The Novel dye composition as claimed in any one of claims 1 or 3, wherein the amount of lemon juice present in the range of 40 % v/v. 5. The Novel dye composition as claimed in claim 1, wherein the source of ammonia is selected from the group comprising ammonium hydroxide, liquid ammonia and ammonium sulphate. 6. The Novel dye composition as claimed in claim 5, wherein the source of ammonia is preferably liquid ammonia. 7. The Novel dye composition as claimed in any one of claims 1 or 6, wherein the amount of liquid ammonia present in the range of 20 % v/v. 8. The Novel dye composition as claimed in claim 1, wherein the alcohol is selected from the group comprising methanol, ethanol, propanol and butanol. 9. The Novel dye composition as claimed in claim 8, wherein the alcohol is preferably ethanol. 10. The Novel dye composition as claimed in any one of claims 1 or 9, wherein the amount of ethanol present in the range of 20 % v/v. 11. The Novel dye composition as claimed in claim 1, wherein the source of sugar is a selected from the group comprising glucose, sucrose, maltose and lactose. 12. The Novel dye composition as claimed in claim 11, wherein the source of sugar is preferably sucrose. 13. The Novel dye composition as claimed in any one of claims 1 or 12, wherein the source of sugar is present in a quantity is in the range of 12 to 20 % w/v. 14. The Novel dye composition as claimed in claim 13, wherein the quantity of sugar present is in the range of 16% w/v. 15. The Novel dye composition as claimed in claim 1, wherein the plant parts of Lawsonia inermis are selected from the group comprising leaves and tender stem. 16. The Novel dye composition as claimed in claim 1, wherein the petroleum based paste or extract is selected from the group comprising petrol based extract or petrol based paste. 17. A process for preparing the novel dye composition as claimed in claim 1, said process comprising the steps of: (a) stored lawsonia inermis plant part is ground; (b) Adding and mixing petroleum solvent with the ground lawsonia inermis plant part of step (a) to obtain a petroleum based paste or a petroleum based extract; (c) adding to the petroleum based paste or the petroleum based extract thus obtained in step (b) 35 to 45% v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia, 17 to 23 % v/v of alcohol and optionally a source of sugar to obtain a mixture; and (d) allowing the mixture obtained in step (c) to stand for a time period in the range of 20 to 28 hours and decanting the same to obtain the dye composition. 18. The process as claimed in claim 17, wherein the plant parts of Lawsonia inermis are selected from the group comprising leaves and tender stem. 19. The process as claimed in claim 17, wherein the petroleum based extract /paste is selected from the group comprising petrol based extract or petrol based paste. 20. The process as claimed in claim 17 wherein in step (a) is 120 gms of wet Lawsonia inermis leaves stored at -20°C is ground and is 20 ml of petrol is added to the same and thoroughly mixed to obtain petrol based paste. 21. The process as claimed in claim 17 wherein in step (b), the acid is selected from the group comprising lemon juice, galgal/hill lemon juice and lime juice. 22. The process as claimed in claim 21, wherein the acid is lemon juice. 23. The process as claimed in any one of claims 17 or 22, wherein the amount of lemon juice added is preferably 25 ml. 24. The process as claimed in claim 17 wherein in step (b), the source of ammonia is selected from the group comprising ammonium hydroxide, liquid ammonia and ammonium sulphate. 25. The process as claimed in claim 24, wherein the source of ammonia is liquid ammonia. 26. The process as claimed in any one of claims 17 or 25, wherein the amount of liquid ammonia added is preferably 12.5 ml. 27. The process as claimed in claim 17 wherein in step (b), the alcohol added is selected from the group comprising methanol, ethanol, propanol and butanol. 28. The process as claimed in claim 27, wherein the alcohol is ethanol. 29. The process as claimed in any one of claims 17 or 28, wherein the amount of ethanol added is preferably 12.5 ml. 30. The process as claimed in claim 17 wherein in step (b), the source of sugar added is a selected from the group comprising glucose, sucrose, maltose and lactose. 31. The process as claimed in claim 17, wherein the source of sugar added is preferably sucrose. 32. The process as claimed in any one of claims 17 or 31, wherein the quantity of sugar added is in the range of 12 to 20 % w/v. 33. The process as claimed in claim 32, wherein the quantity of sugar added is preferably 10 gms. 34. A method for conducting sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay by using the novel dye composition as claimed in claim 1, said method comprising the steps of: (a) preparing denatured protein sample; (b) preparing polyacrylamide gel; (c) loading the denatured protein sample on to the polyacrylamide gel and applying electric field across the same; (d) staining the polyacrylamide gel of step (c) using the novel dye composition comprising 15 to 25 % v/v of petroleum based paste or extract of the plant parts of Lawsonia inermis, 35 to 45 % v/v of an acid having pH in the range of 2 to 3, 17 to 23 % v/v of source of ammonia and 17 to 23 % v/v of alcohol and optionally a source of sugar to obtain stained polyacrylamide gel; and (e) de-staining the stained polyacrylamide gel. 35. The method as claimed in claim 34 wherein in step (c), the polyacrylamide gel is stained using the novel dye composition for a time period in the range of 30 to 35 minutes at temperature in the range of 40° to 45°C. 36. The method as claimed in claim 34, wherein the step of de-staining the stained polyacrylamide gel comprises: (i) treating the stained polyacrylamide gel with acetic acid having a first concentration level for a first time period to obtain a partially de-stained polyacrylamide gel; and (ii) treating the partially de-stained polyacrylamide gel thus obtained with acetic acid having a second concentration level for a second time period to obtain substantially de-stained polyacrylamide gel. 37. The method as claimed in claim 36, wherein the first concentration level of acetic acid is in the range of 45 to 55%. 38. The method as claimed in claim 37, wherein the first concentration level of acetic acid is preferably 50%. 39. The method as claimed in claim 36, wherein the value of the first time period is in the range of 20 to 35 minutes. 40. The method as claimed in claim 39, wherein the value of the first time period is preferably in the range of 25 to 30 minutes. 41. The method as claimed in claim 36, wherein the second concentration level of acetic acid is in the range of 5 to 10 %. 42. The method as claimed in claim 41, wherein the second concentration level of acetic acid is preferably 7%. 43. The method as claimed in claim 36, wherein the value second time period is in the range of 70 to 100 minutes. 44. The method as claimed in claim 43, wherein the value second time period is preferably in the range of 80 to 90 minutes. 45. The method as claimed in claim 36, wherein step (ii) is performed at elevated temperature in the range of 65 °C to 70°C. 46. A dye composition prepared from plant parts of Lawsonia inermis substantially as herein described with reference to the foregoing example and the accompanying drawings.

Documents:

1216-DEL-2007-Abstract-(14-11-2011).pdf

1216-del-2007-abstract.pdf

1216-DEL-2007-Claims-(14-11-2011).pdf

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1216-DEL-2007-Correspondence Others-(14-11-2011).pdf

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1216-DEL-2007-Description (Complete)-(14-11-2011).pdf

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1216-del-2007-form-2.pdf

1216-DEL-2007-Form-3-(14-11-2011).pdf

1216-del-2007-form-3.pdf

1216-DEL-2007-Form-5-(14-11-2011).pdf

1216-del-2007-form-5.pdf

1216-DEL-2007-GPA-(14-11-2011).pdf