FIELD OF THE INVENTION

The present method relates to an apparatus for staining and immunolabeling of biomolecules on gels, membranes, slides or similar surface in cool mist phase. In particular, the present invention provides an automated system, which uses mist phase in place of liquid solutions for incubating gels or membranes or slides, which in turn reduces background interferences due to less non specific binding, performs without the need of robotics and exhibits improved level of detection. The invention is immensely useful in the field of biochemistry and molecular biology and specifically for the detection of proteins using antibodies with methods like immunoblotting, immunohistochemistry and electron microscopy. The system can perform automated immunoblotting, immunohistochemistry, Coomassie Brilliant Blue staining, silver staining and similar methods. The developed apparatus finds application in the field of diagnostics, biotechnology, food, drugs & pharmaceuticals.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF PRIOR ART

Detection and quantification of proteins is widely and commonly used technique for most of the researchers. Levels of proteins can be detected in situ in the tissue or cells using immunohistochemistry or immunocytochemistry and in the cellular or tissue lysates using Immunoblotting (Western blotting). Immunohistochemistry and immunocytochemistry are the most commonly used in situ detection methods, while for subcellular localization electron microscopy is used. For quantification of specific proteins in tissue or cell lysates, immunoblotting is most commonly used method. Immunoblotting is the electrophoretic parting of proteins, which are further transferred from the gel to a solid support and detected via epitope specific immune reaction. The process comprises of electrophoresis, electro-transfer and immunological signal detection.

All the above-mentioned methods serve a great role in research setup, however their translation in diagnostics is still a challenge, mainly due to multiple steps, requirement of highly skilled person and variability in results while repeating the experiments. In both immunohistochemistry and immunoblotting, soaking of the samples is required for incubating them with antibodies or blocking reagent.

Detection of proteins using electrophoresis was discovered in 1979 by Towbin et. ah, (PNAS, 1979. 76:4350-4354). In 1981, the method was improved and popularized in name of Western blotting by Burnette WN (Analytical Biochemistry, 1981. 112:195-203). Later on the method has become the gold standard, which involves the following steps: a) Lysis of tissue or cell samples and preparation of protein samples in Laemmli buffer. b) Separation of proteins according to their molecular weights on SDS-PAGE. c) Transfer of separated proteins on nitrocellulose or PVDF membrane. d) Blocking of free sites of membrane using non-fat milk or Bovine Serum Albumin (BSA). e) Incubation with primary antibody specific for target protein and washing of unbound antibody. f) Incubation with labeled secondary antibody specific for primary antibody and washing of unbound secondary antibody. g) Detection and quantification of target protein by signal generated from labeled secondary antibody chemiluminescence, fluorescence and colorimetric are three major types of detection methods used.

In above protocol, steps [d] to [g] are carried out manually and require incubation of membranes in different reagents in liquid phase with shaking. Multiple washings required in between blocking and incubation of primary and secondary antibody makes the process tedious. In the recent past, new methods have been developed which use sequential lateral flow technology (Thermo Scientific) or vacuum driven flow of antibodies through membrane (SNAP I.D). However, both the methods are not cost effective and still use antibody and blocking reagents in liquid form. One more capillary-based electrophoresis method has been developed, which requires huge instrument and is gel free and blot free, which means blot image is artificially developed based on the detection of proteins in capillary electrophoresis (Nature Methods; 2011, 8, page 982). Atomizers are known and used majorly as humidifiers in rooms or offices, but have not been reported or conceived till date for performing automated immunoassays for detecting proteins and antibodies. Conventionally immunoblotting and gel/ membrane staining is done manually by incubating gels or membrane in liquid phase and keeping them on shaker and changing different reagents manually and none of the reported systems for the same are automated while using cool mist phase for all the steps of the process.

Accordingly, keeping in view the drawbacks of the hitherto reported prior art, the inventors of the present invention realized that there exists a dire need to provide an automated system/ apparatus for immunoassays wherein the conventional atomizers can be improvised so as to make them suitable for detecting biomolecules in membranes or slides; by adding a timer facility, which can count the running time of each mist generator; adding a membrane/ slide holder box or plate, which is used to hold the membrane or slide; combining multiple atomizers in such a way that they can run in the relay with the timing facility to generate cool mist from different reservoirs/ tube as per specified; and an absorbent stick is placed below each atomizer disc, which is required for continuous flow of different solutions from reservoirs to disc and wherein the system is automated without the need of robotics, generates images with low background and does not over-stains gel or membranes, is less labor oriented, does not require highly skilled manpower and is thus cost effective.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is therefore to provide an apparatus for staining and immunolabeling of biomolecules on gels, membranes, slides or similar surface in cool mist phase which obviates the drawbacks of the hitherto reported prior art.

Another objective of the present invention is to provide an apparatus for the detection of proteins or biomolecules, wherein several steps are automated thereby reducing the manual differences or errors between different assays and improving the quality of detection.

Still another objective of the present invention is to provide an apparatus, which uses cool mist in place of liquid soaking, making it easy to automate the process steps with reduced variability and increased quality of detection. Yet another objective of the present invention is to provide an apparatus which is automated without the need of robotics, generates images with low background and does not over-stains gel or membranes, is less labor oriented, does not require highly skilled manpower and is thus cost effective.

Still another objective of the present invention is to provide a staining technique that stains and/or destains proteins in a more effectual way and reduces the problem of overstaining of gel and/or membrane.

SUMMARY OF THE INVENTION

The present invention provides a novel apparatus, which is an automatic system for detecting protein-protein or protein-nucleic acid interaction or simple protein staining on different kind of matrices. The developed system involves the use of antibodies or other biomolecules in cool mist form in place of liquid for detecting levels of proteins or nucleic acids in different kind of setups like immunoblotting, immunohistochemistry, gel labelling, electron microscopy and the like.

Atomizers are well known in the art and have been used for a wide variety of purposes such as humidifiers in rooms or offices; spraying of oils, paints, fertilizers, and other liquids; disinfecting equipment; humidification in industrial process control; and for a therapeutic purpose to treat respiratory diseases. However, the apparatus developed in the present invention has been invented for using it in biomolecular interaction studies mainly proteins and antibodies. Following additions in conventional atomizers makes them useful for detecting biomolecules in membranes or slides: a) Adding a timer facility, which can count the running time of each mist generator. b) Adding a membrane/ slide holder box or plate, which is used to hold the membrane or slide. c) Combining multiple atomizers in such a way that they can run in the relay with the timing facility to generate cool mist from different reservoirs/ tubes as per specified. d) An absorbent stick is placed below each atomizer disc, which is required for continuous flow of different solutions from reservoirs to disc. e) A single atomizers connected to multiple reservoirs. In an embodiment, the present invention provides an improved technique of staining proteins immobilized in and/or on a solid matrix (such as a gel or porous particle) or solid support (such as a membrane or porous filter); and improved immunodetection of the membrane and/or tissue section.

In another embodiment, the present invention provides a staining technique that stains and/or destains proteins in a more effectual way and reduces the problem of overstaining of gel and/or membrane.

In still another embodiment, the present invention provides an automated method of staining gel and/or membrane, and immunolabeling of membrane and/or tissue sections; consequently increasing the reproducibility of results and reducing variability due to multiple manual steps.

In yet another embodiment, the present invention provides a method of staining and immunolabeling proteins using solutions in a cool-mist form which reduces gel overstaining and increases the antigen-antibody and/or antibody-antibody contact without using hefty shakers and robotics.

In still another embodiment, the present invention avoids the loss of specimen from the IHC slide and prevents the problem of sections drying out. The present invention thus provides an automated method that consistently and reproducibly stains proteins immobilized in a gel/bead (polyacrylamide or agarose), on and/or in a membrane/fiber filter; and immunodetection of membranes and/or tissue sections using solutions in the cool mist phase. All gel/membrane staining and membrane/tissue immunolabeling steps (i.e., blocking, washing, and primary and secondary antibody incubations) of western blotting and/or IHC can be efficiently performed with this cool mist system. All blocking, antibody incubation and washing steps are hands-free and thus greatly reducing the variability of results due to manual processing. This utility model has substantially decreased overstaining of the gel and/or membrane and increased local antibody concentrations at binding sites driving antigen-antibody/antibody-antibody binding reaction forward without the employment of hefty shakers. The present system is compatible with all known methods of detection like fluorescent, chemiluminescent, Infra-red fluorescent, or chromogenic detection. The conventional western blotting is manually intensive and time-consuming that relies on the slow diffusion of reagents into and out of the blot in a shaking state which increases the chance of productive collisions on the solid phase, leading to long incubation times and possible high background. Efforts have been made to improve western blotting by reducing time and reagent requirements and introduce automation of manual steps. The present invention provides an automated method of blot processing using blocking, antibody solutions, color development solutions and washing reagents in the mist form. Traditionally, all the laboratories carry out an antibody incubation step in plastic boxes of varying sizes. Incubation carried out in boxes helps antibody solution to spread with uniformity throughout the film surface. However, size of membranes varies according to number of samples required for different studies. Many times, larger volume of antibody solution is used due to unavailability of perfect sized boxes. Antibody incubation steps of western blotting using antibody solutions in the mist form would allow efficient collisions between the protein of interest and antibody solutions employing membranes of varied dimensions without compromising the quality of results.

In contrast to conventional western blotting technique, where diffusion is the principal means of transport of reagents, the present mist driven system uses an ultrasonic mist maker to actively expose the gel/membrane with staining, destaining, blocking, washing, and antibody solutions. The ultrasonic humidifier used in the present invention consists of a piezoelectric ceramic disc. This ceramic disc converts high-frequency electrical signal that generally has a resonating frequency of around 113 KHz into high-frequency mechanical vibration. Ultrasonic waves are converged on to the solution; when the solution can no longer follow the high-frequency vibrations of the ultrasonic transducer, a transitory vacuum is formed on the negative oscillation of the transducer where the solution cavitates into mist at the spray surface of the disc. Staining/destaining solutions, blocking solution, wash buffers, and antibody solutions are loaded in the tube/tubes placed below the ultrasonic humidifier disc/discs as per their required dilution and amount. The pre-wet gel/membrane with immobilized proteins is placed in the box with its protein side down. The gel/membrane is exposed to the mist of the desired solution for the specific period of time as defined by the single tube mist system/ four-channel relay module. The stained gel/membrane can be viewed visually or by using UV or blue/green-light transilluminators or imaging instruments equipped with suitable filters. Membrane/IHC slide can be developed by selecting a desirable technology (like chemiluminescence, fluorescence, or chromogenic) depending on the type of secondary antibody employed and the instrument available.

The steps of the present embodiment of invention do not differ from those of a conventional manual method of IHC. Particularly, the reagents and their order of addition and incubation times and temperatures do not vary significantly. However, the form in which the blocking/ antibody/wash solutions are applied to and incubated with the tissue section is in the cool mist phase and is fully automated. Since numerous washing steps are suggested in between each incubation step, high care must be exercised to circumvent the loss of tissue sections from the slide. Moreover, very thorough washing of the tissue specimen between antibody applications is vital particularly to remove any unbound antibody which would otherwise result in background signals. Excess liquid must also be removed by means of allowing it to drip off the slide or by placing the edge of the slide on the blotting paper to prevent any dilution of the antibody solutions, yet sections must on no account be allowed to dry out. Enough antibody solutions must be applied to fully cover the slide area where the assay region may occur, but waste should be kept to the absolute minimum. The present invention solves these problems by reducing the risk of tissue sections fall off as slides are just put in the box with assay region exposed to the mist of appropriate solutions, without any dripping or blotting steps needed for excess solution removal. Solutions in cool-mist phase have increased antigen-antibody and/or antibody-antibody contact which would otherwise be low in the conventional method where antibody solutions are applied on to the tissue section in the form of drops.

In an embodiment, the present invention provides an apparatus wherein, the inventors have automated the steps and multiple washings as mentioned above by using automatic cool mist generated from ultrasonic humidifier and which replaces the incubation of membrane in liquid with mist. The claimed method keeps the practice and simplicity of using gels and membrane and does not requires any new reagent or consumable. As this method does not require submerging of the membrane in liquid, over binding of antibodies is reduced. Moreover, automation of all the incubation steps substantially reduces the variability between different detections and makes it more user friendly. In a further embodiment, the present invention provides an apparatus for staining and immunolabeling of biomolecules on gels, membranes, slides or similar surface comprising a box for holding membrane or gel (1), an absorbent stick (2) and tube (3) for holding reagent or reagents; an ultrasonic atomizing ceramic disc (9) attached to an absorbent stick (2) dipped in reagents holding reservoir (3); a timer facility and a DC power source (13); a box on top of incoming mist to hold the gels/ samples (1) power driver boards forming a 4-channel relay module controlled by the microcontroller (16) that acts as controller for the complete system and provides inputs to the relay module (17); a DC current power source providing power for operating the system; wherein the power of transducer or mist humidifier plates (18, 19, 20, 21) is controlled through the 4-Channel relay module (17), being controlled by the microcontroller (16); wherein the micro controller counts the running time of each disc and provides high low voltage to relay board to control the power of each disc. A power driver board is used, which contains wires (4), terminal (5), start/stop switch (6) and Power interface (7); a membrane (8), which contains proteins separated via conventional SDS-PAGE along with protein ladder placed on top of ultrasonic mist maker fogger ceramic disc (9), where a cool mist (10) is generated.

In still another embodiment, the present invention provides an apparatus, applicable for automatic staining, destaining, immunoblotting, immunohistochemistry, immunocytochemistry, protein labeling in electron microscopy, immunoprecipitation and other similar biomolecule detection methods.

In yet another embodiment, the present invention provides an apparatus, wherein the gel or membrane or slide is exposed to mist generated by conventional atomizers or electrostatic atomization mechanisms.

In still another embodiment, the present invention provides an apparatus, wherein a box holding membrane or gel (1) is separated from mist generator (9) and connected with mist generator through pipe.

In yet another embodiment, the present invention provides an apparatus, wherein it contains two or more than two reservoirs (3), each with separate atomizer with absorbent stick (2) placed below it, automatic programmable system for generating cool mist of antibody (Fig.7), blocking and washing solutions passing through different reservoirs (3) in sequence as required, a box for holding gel or membranes or immunohistochemistry slide (1, 14), and a timer circuit for programming (Fig. 7).

In still another embodiment, the present invention provides an apparatus, wherein the samples or gels are selected from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, or 2-Dimensional SDS-PAGE gels, or membranes or native SDS-PAGE gels or gradient SDS-PAGE gels, or slides containing tissue sections.

In yet another embodiment, the present invention provides an apparatus, wherein it has four reservoirs or tubes (3) each containing four different or same solutions of antibodies or reagents required for immunoblotting and automated to run the different steps including washing, blocking, primary antibody incubation, secondary antibody incubation of immunoblotting; wherein one or more reservoir or tube holds solution for washing the membrane; one or more reservoir or tube holds solution for blocking the non-specific sites on membrane; one or more reservoir or tube holds solution for primary antibody incubation; one or more reservoir or tube holds solution for secondary antibody incubation; and a control system programmed for making cool mist from different reservoirs as per required sequence and time.

In still another embodiment, the present invention provides an apparatus, wherein the reservoirs (3) hold dyes selected from coomassie blue or silver stain or 5-bromo-4-chloro-3-indolyl- phosphate/nitro blue tetrazolium (BCIP/NBT).

In yet another embodiment, the present invention provides an apparatus, wherein mist is generated through one or more than one atomizer (9) and passed through plurality of small pores or holes to membrane or gels or slides (1) for equal distribution.

In another embodiment, the present invention provides a process for staining and immunolabeling of biomolecules on gels, membranes, slides or similar surface in cool mist phase setup using the developed apparatus comprising the steps of washing, blocking and primary and secondary antibody incubation steps of Western blotting in automatic way using four tubes (3) attached with machine for four different solutions selected from washing solution (TO), blocking solution (Tl), primary antibody solution (T2), and secondary antibody solution (T3); four ultrasonic ceramic discs (DO, Dl, D2, D3) placed on said four tubes (TO, Tl, T2, T3) containing different solutions and absorbent sticks as illustrated in Figure 4; wherein the power of ultrasonic ceramic discs (9) is controlled through the power driver boards (17) forming a 4-channel relay module being controlled by the microcontroller (16), all enclosed in a separate box (12) as illustrated in Figure 5 and wherein the power supply for operating the system is a DC current power source (13).

BRIEF DESCRIPTION OF THE ACCOMPANYINGFIGURES

In the figures accompanying the specification;

Figure 1 represents the front view of the single tube mist system for gel and membrane staining. Our single tube mist system comprises of a tube (3) fitted in a stand with its mouth opened in a box where gel/membrane/slide with tissue section (1) is placed with its protein/tissue section side down. The cotton absorbent stick (2) is fitted at the center of the tube and the tube is filled with different solutions like staining/detaining solution for gel/membrane staining or destaining or blocking solution/washing solution/primary antibody solution/secondary antibody solution for western blotting or IHC. An ultrasonic ceramic disc (9) is placed at the mouth of the tube which convert the solution into mist (10). The ultrasonic ceramic disc is connected to a power driver board through wires (4) at the terminal (5). The power driver board is equipped with start/stop switch (6) and power interface (7). The solution in mist form strikes the gel/membrane/slide surface and condenses, consequently carrying out staining or labeling of biomolecules in the cool mist phase as shown in Figure 1.

Figure 2 represents Side view of 4-tubemist system for automated Western blotting. /IHC. The 4-tube mist system used to carry out immunolabeling of biomolecules on a membrane/tissue section (1) comprises of 4 tubes (TO, Tl, T2, T3) (3) fitted in a box keeping their mouth slightly tilted towards the center of the box cap where the membrane/slide with tissue section is placed. All the 4 tubes are provided with cotton absorbent sticks (2) which help in the movement of solution to the discs where the mist is generated. For carrying out western blotting/IHC in a cool mist phase, these 4 tubes are filled with different solutions like washing solution (TO), blocking solution (Tl), primary antibody solution (T2), and secondary antibody solution (T3). The box shown in the Figure 2 is properly closed during the operation to avoid any leakage of mist. Figure 3 represents detailed sectional view of the ultrasonic atomizing ceramic disc along with power driver board. The ceramic disc (9) has a diameter of 20 mm with the resonating frequency of around 113 KHz. The backside of disc atomizer (11) is made up of ceramic, a piezoelectric material, which when exposed to electric current that matches the natural frequency of the piezoelectric material oscillates at an ultrasonic frequency. The high frequency sound waves formed are transposed into mechanical energy that is transferred to the solution, generating standing waves. When the solution can no longer follow the high-frequency movement of the oscillating disc, a momentary vacuum is created and the solution exits the atomizing surface of the disc (10) as mist of uniform micron-sized droplets. The disc is connected to a power driver board through wires (4) at the terminal (5) which is equipped with Start /stop switch (6) and Power interface (7) as shown in the figure.

Figure 4 represents detailed top view of the 4-tube mist system used in the invention. Four ultrasonic atomizing ceramic discs (DO, Dl, D2, D3) (9) are placed on four tubes (TO, Tl, T2, T3) containing different solutions and absorbent sticks. All four ultrasonic ceramic discs are connected to four power driver boards with wires. The pre-wet membrane (1) which contains protein separated by conventional SDS-PAGE along with protein ladder (8) is placed on the cap of the box and exposed to mist of different solution as defined by 4-channel relay module. During the operation, the solutions leave the atomizing surface of the discs (10) as mist and strike the membrane and condense, thus carrying out labeling of biomolecules in the cool mist phase.

Figure 5 represents top view of the complete setup for 4-tube mist system. The right box shown in the figure is equipped with 4-tubes (TO, Tl, T2, T3) containing different solutions and absorbent sticks. Four ultrasonic atomizing ceramic discs (DO, Dl, D2, D3) connected with power driver boards are placed on each tube for mist generation. The pre-wet membrane/slide with tissue section (1) is carefully placed in the box which is exposed to mist as per the defined program. The left box (12) shown in the figure contains power driver boards connected to four discs, microcontroller, adaptors, and switches used to generate the electronic setup of the present invention. A DC current power source (13) is used to supply power for operating the system at an appropriate voltage and current. Figure 6 represents detailed top view of 4-tube mist system used in immunohistochemistry. (IHC). The setup comprises of four tubes (TO, Tl, T2, T3) fitted in box where the slide (14) containing tissue section (15) is placed. Four cotton absorbent sticks are placed at the centre of tubes and the tubes are filled with different solutions like blocking solution/washing solution/primary antibody solution/secondary antibody solution used in IHC. Four ultrasonic ceramic discs ((DO, Dl, D2, D3) (9) connected with power driver boards through wires are placed on the mouth of each tube for mist generation at the atomizing surface of the discs (10). During operation, the box is carefully closed with slide containing tissue section facing the ultrasonic ceramic discs. The tissue section is exposed to different solution in a cool mist phase as per the relay described in the illustrated example of the present invention. The box used in the invention must be leak proof to avoid any leakage of mist which might reduce the efficiency of the process.

Figure 7 represents detailed electronic circuit of the 4-tube mist system. The electronic circuit of the present invention comprises of a microcontroller (Arduino Uno) (16) which acts as controller for the complete system and provides inputs to the relay module (17). The microcontroller count the running time of each ultrasonic ceramic disc and provides high low voltage to relay board to control the power of each disc, thus controlling the exposure time the gel/membrane/slide is exposed to the mist of different solutions. The power of transducer or ultrasonic ceramic discs (18, 19, 20, 21) is controlled through the 4-Channel relay module (17), which is being controlled by the microcontroller (16). The DC current power source supply power for operating the whole system.

Figure 8 represents the Flow Chart describing the differences between the method of staining using the conventional methods and the apparatus developed in the present invention. The membrane with tranferred proteins/silane coated slides with tissue section are processed employing both conventional method and automated mist-based method. In the conventional method, the membrane/slide is transferred to a box containing a desired blocking solution and incubated at RT for 1 hour on a shaker. The blocking solution is then carefully removed by draining and the membrane/slide with tissue section is incubated with an appropriate primary antibody solution against the protein of interest for 3 hours at RT on a shaker. Any unbound and loosely bound primary antibody is removed by washing the membrane/slide three times for 5 minutes with IX TBST. The primary antibody binding is then detected by a method that typically involve the application of secondary antibody that is raised against the primary antibody, and to which a fluorophore or an enzyme has been covalently attached. The unbound secondary antibody is removed by washing membrane/slide with tissue section three times for 10 minutes with IX TBST. The bound antibodies are then detected using an appropriate detection system. LICOR-Odyssey Infrared Imaging System has been used to detect IR-Dye tagged secondary antibodies in the illustrated examples of the present invention. Alexa Fluor 488 secondary antibodies has been employed in IHC and imaging is done using EVOS® FL Auto Imaging System. Alternatively, in our newly developed system, all these steps (blocking, washing, primary and secondary antibody incubation) are automated. Membrane/slide containing tissue section is just placed in a box equipped with ultrasonic ceramic discs. The membrane/slide containing tissue is exposed to solution in a cool mist phase as per the defined relay module. Membrane/slide containing tissue section is then developed/imaged using an appropriate detection method.

In a further embodiment of the present invention, for staining and destaining gel that run on an SDS-PAGE following standard protocol, both conventional and automated mist-based methods have been employed. Conventional gel staining procedures involve submerging the fixed gel in a coomassie staining solution and putting it on shaker for 1 hour at RT. After 1 hour of staining, gel is destained with destaining solution with replenishing the solution several times until the background of the gel becomes clear. It takes around 90 minutes to effectively destain the gel. Imaging is done using an appropriate imaging system. Alternatively, in the developed system, the gel is transfered to a box and exposed with the mist of coomassie staining solution for 1 hour. The gel stained with cool mist system just takes 45 min to effectively get destain and also shows better intensity of bands as compared to bands obtained using conventional method. Imaging of gel is done using an appropriate imaging system. The G-800 Calibrated Densitometer (BioRad) has been used for imaging gels in the illutrated examples of the present ivention. DETAILED DESCRIPTION OF THE PRESENT INVENTION

The terms “apparatus” and “system” are used interchangeably throughout the specification.

Referring to the figures in detail, the apparatus of the present invention comprises a box for holding membrane or gel (1), an absorbent stick (2) and tube (3) for holding different solutions like staining/detaining solution or blocking solution or washing solution or primary antibody solution or secondary antibody solution. A power driver board is used, which contains wires (4), terminal (5), start/stop switch (6) and Power interface (7). A membrane (8), which contains proteins separated via conventional SDS-PAGE along with protein ladder placed on top of ultrasonic mist maker fogger ceramic disc (9), where a cool mist (10) is generated.

The backside of disc atomizer (11) is a piezoelectric material as illustrated in Figure 3. In general, piezoelectric materials display a unique range of characteristics. In the most basic sense, if a piezoelectric material is compressed, the material becomes electrically charged and an electric current is produced. The greater the pressure, the greater will be the electric current. If the material is all of a sudden stretched rather than being compressed, the direction of the current will be reversed. This is referred to as the piezoelectric effect. The opposite of this occurrence also holds true: It follows that with the application of an electric current that matches the natural frequency (also known as eigenfrequency) of the piezoelectric material, the material can be made to expand and contract; and ultrasonic waves are produced. This phenomenon is known as the inverse piezoelectric effect. The piezoelectric element is often clad with stainless steel for erosion resistance. In the present invention, the piezoelectric ceramic disc is used, and it oscillates in the presence of electrical current at an ultrasonic frequency. As the speed of oscillation is increased to a point where the particles of water can no longer follow the oscillating surface, a transitory vacuum and high compression occur. This area of low pressure is known as the cavity. At cavitation, broken capillary waves are generated, and consequently at the crest of the wave, extremely minute droplets of water possess adequate amount of energy to break up the surface tension and move out of water. The moment these minute droplets of water exit the surface of water they are evaporated into the airflow and leave the humidifier as a fine mist of uniform micron-sized droplets. The size of these aerosol particles would be smaller with increasing transducer frequency. The solution in mist form strikes the gel/membrane/slide surface and condenses, thus carrying out staining or labeling of biomolecules in the cool mist phase as illustrated in Figure 1.

The single-tube mist system for gel/membrane staining comprises of a tube containing solution held on a stand and fitted in a box with its mouth facing the box. Ultrasonic mist maker fogger ceramic disc (9) is placed on the mouth of the tube containing different solutions and absorbent stick (2) as shown in Figure 1. The cotton absorbent stick (2) is well structured in bright white colour and has good absorbability and elasticity, big water storage capacity, stable volatility, and is non-sticky. The length of the absorbent stick is cut to match the length of the tube containing solution; and in order to ensure stable mist generation, the cut one end is dipped in the solution and the original one is put in contact with the disc atomizer. An automated 4-tube mist system employed to carry out immunoblotting is illustrated in the Figures 2, 3 and 5. The setup is designed to carry out washing, blocking and, primary and secondary antibody incubation steps of Western blotting in automatic way using four tubes (3) attached with machine for four different solutions like washing solution (TO), blocking solution (Tl), primary antibody solution (T2), and secondary antibody solution (T3). Four 20mm Ultrasonic ceramic discs (DO, Dl, D2, D3) are placed on four tubes (TO, Tl, T2, T3) containing different solutions and absorbent sticks as shown in Figure 4. The power of ultrasonic ceramic discs is controlled through the power driver boards forming a 4-channel relay module which is controlled by the microcontroller (Arduino Uno), all enclosed in a separate box (12) as illustrated in Figure 5. The power supply for operating the system is a DC current power source (13). The embodiment of the invention illustrated in Figure 6 is identical to the embodiment illustrated in Figure 1-5 with the exception of a silane coated slide (14) containing tissue section (15) in place of gel/membrane to carry out immunohistochemical staining.

Figure 7 illustrates the detailed electronic circuit of the 4-tube mist system. According to an embodiment of the invention, the device includes a timer facility to count the running time of each disc and control the exposure. The time duration of each disc can be controlled either through the microcontroller program or through the mobile app. In the proposed system a microcontroller (Arduino Uno) (16) acts as controller for the complete system and provides inputs to the relay module (17). A DC current power source provides power for operating the system. The power of transducer or mist humidifier plates (18, 19, 20, 21) can be controlled through the 4-Channel relay module (17), which is being controlled by the microcontroller (16). Micro controller counts the running time of each disc and provides high low voltage to relay board to control the power of each disc.

Though the preferred embodiment of the device has been described in detailed but it must be understood that within the scope of this invention various changes may be made in the form, details, and arrangement of parts, the combination thereof and mode of operation, which in general described consist in a device capable of carrying out the objects set forth, as disclosed and defined in the appended claims.

METHODS

To check the functionality of the claimed device, protein samples on PVDF or nitrocellulose membrane (immunoblotting), tissue sections on slides or protein samples on SDS-PAGE gels (Coomassie Blue Staining) etc were taken. Protein samples were prepared using RIPA buffer [(pH 7.6); containing 50 mM Tris-HCl, 150mM Sodium chloride, 2mM EDTA, 2mM EGTA, 1% v/v Triton X-100, 10 mM Sodium fluoride, 2mM Sodium orthovanadate, 1% w/v Sodium deoxycholate, 1% w/v Sodium dodecyl sulphate, lmM PMSF and protease inhibitor cocktail] as it is regularly carried out in our laboratory using following method. 500 mΐ RIPA buffer containing an appropriate amount of protease inhibitor cocktail and dithretiol (DTT) was used for every 10 mg of tissue. Tissue homogenate was thoroughly incubated on ice for 30 minutes and vortexed occasionally. After incubation, the homogenate was centrifuged at 10,000g for 20 minutes at 4°C. The clear supernatant was then transferred to a fresh tube and the pellet was discarded. The protein concentration of the lysate was determined via bicinchoninic acid (BCA) protein estimation assay. Samples were stored at -80 °C for longer use. The method of this invention is not limited to the isolation of protein from the brain using RIPA method and its quantification via BCA method, which is one suitable utility thereof. The samples may be any biological sample containing protein; and the protein can be isolated by employing any suitable protein isolation protocol. The sample could be a tissue biopsy, blood, blood plasma, cultured cells, extracellular fluid, culture media, plant matter, synthetic proteins, archaeal, bacterial and/or viral samples, or fungal tissue. The sample may contain intact proteins, fully/partially denatured proteins, protein fragments, or partially degraded proteins. Alternatively, the total protein concentration of samples can be determined using Bradford (Coomassie), Lowry, and other suitable protein estimation assays.

In the illustrative examples of the present invention, protein samples after quantification with BCA method were denatured in 2X Laemmli buffer (1 : 1 ) for 10 minutes at 95 °C and then chilled on ice. By heating the protein samples between 70-100°C in the presence of excess SDS (a detergent) and P-mercaptoethanol/ Dithiothreitol or DTT (a reducing agent) as present in Laemmli buffer, both intra and inter-molecular disulfide bonds are reduced, and the protein is fully denatured and dissociated into its constituent subunits. Any method of denaturing proteins that is unlikely to cause chemical modifications may be preferred. In the present invention, the protein samples prepared in Laemmli buffer were then subjected to SDS-PAGE (stacking gel: 5% acrylamide and separating gel: 10% acrylamide). PAGE may be carried out with gels of different sizes, various concentrations of polyacrylamide, and ratios of acrylamide monomer to bis-acrylamide crosslinker, with number of different electrophoretic buffer systems, and in the presence or absence of SDS. After electrophoresis, proteins can be detected in a gel using different protein staining procedures, transferred onto the surface of membrane for detection by blotting methods and/or undergo excision and extraction for mass spectrometric analysis.

Coomassie Blue Staining: Any gel wherein proteins have been separated by electrophoresis can be stained with the present invention following standard staining protocols. Protein samples along with 3m1 prestained protein ladder were run on 10% SDS-PAGE gel and were stained using Coomassie blue stain (R250).The invention can also be used to stain gels where the proteins have been separated by a technique known as isoelectric focusing used for 2-D gels. Alternatively, other stains such as Sypro Ruby, Silver nitrate, Coomassie dyes (G250), Larva purple, Flamingo, Stains-all, Deep purple, and others might also be used in place of Coomassie blue stain with the present invention. After electrophoresis, the gel was incubated in fixing solution (50% methanol, 10% glacial acetic acid) for one hour with gentle shaking. After one hour, fixing solution was changed and gel was left for overnight in fixing solution with gentle shaking. After overnight incubation, gel was cut into two equal parts. First part was stained using conventional method of incubating in staining solution (0.125% Coomassie Brilliant Blue R-250, 50% methanol, 10% Glacial acetic acid) for 1 hour with gentle shaking, while second gel part was stained for 1 hour using cool mist-based system without any shaking and using same staining solution (0.125% Coomassie Brilliant Blue R-250, 50% methanol, 10% Glacial acetic acid). After 1 hour of staining, gels were destained using destaining solution (40% methanol, 10% glacial acetic acid) with replenishing the solution several times until the background of the gel was cleared. Imaging was done using G-800 calibrated Densitometer (BioRAD). The method of this invention is not limited to stain only gels. It can also be used to stain proteins on and/or in membranes made from natural or artificial sources such as cellulose or its derivatized variants (e.g., nitrocellulose) and nylon or derivatized variants (e.g., PVDF). The stained gel/membrane can be viewed visually and/or imaged using transmissive and reflective imaging to scan chromogenic samples or by using UV or blue/green LED transilluminators or imaging instruments equipped with suitable filters as per the requirement.

Immunoblotting: Protein samples were run on 10% SDS-PAGE gel along with 3m1 prestained protein ladder. Several forms of other polyacrylamide gel electrophoresis (PAGE), viz. Non denaturing PAGE (also called native-PAGE) and Two-dimensional (2D) PAGE can also be employed, and each form providing different types of information regarding proteins of interest. After the completion of the run, proteins from gels were transferred to a PVDF membrane via electroelution (or electrophoretic transfer) wet transfer (or tank transfer) system. Several other electrotransfer strategies may also be used such as semi-dry and dry, each of which need special considerations with regard to time, cost, and required reagents and apparatuses. Alternatively, nitrocellulose membrane might also be used in place of PVDF. Moreover, the proteins may also be applied to the solid support/matrix by other methods such as diffusion transfer, capillary transfer or wicking, vacuum blotting transfer, chromatography, electrophoresis, or electrofocussing.

The membrane supports employed in western blotting possess high affinity for proteins. So, after the proteins has been transferred from the gel, it is vital to block the remaining sites on the membrane to prevent non-specific binding of the detection antibodies/ other detection agents during subsequent steps. An array of blocking reagents that ranges from skimmed milk or normal serum to highly purified proteins (e.g., BSA) have been used to block non-specific binding sites on a membrane. The blocking reagent must improve the sensitivity of western blotting by reducing background noise and improving the signal-to-noise ratio. No single blocking agent is idyllic for every experiment given that each antibody-antigen pair possesses unique characteristics. Empirically testing various blocking buffers to meet experimental requirements is crucial in optimizing a western blot experiment. The transferred membrane in the present invention was blocked with skimmed milk/BSA prepared in IX TBST (50 mM Trizma® base, 150 mM NaCl,0.1% Tween-20, pH 7.5) for 1 hour to avoid any non-specific antibody binding.

After blocking, the membrane was incubated with primary antibody in a suitable bufferraised against the protein of interest. Washing steps are needed to remove unbound antibodies and reduce background, thus obtaining good signal-to-noise ratio. Tris-buffered saline (TBS) and phosphate-buffered saline (PBS) supplemented with 0.05 to 0.5% detergent (like Tween 20) are the most commonly used wash buffers; both can be suitably used with the present invention. Generally, the primary antibody that recognizes the specific target protein in a western blot is not directly detectable. Therefore, the antibody-antigen complex formed on the membrane is then detected by a method that usually involves the application of a secondary antibody, raised against the first (or primary) antibody. These indicator antibodies might be radioactively labeled, luminescent, conjugated with a fluorescent substance (or fluorophore), or conjugated with an enzyme capable of colorigenic reaction on addition of a suitable substrate. The secondary antibody binding step was performed for 1 hour at room temperature. In the present invention, the unbound primary/secondary antibody is washed off with IX TBST after each antibody incubation step leaving only the bound antibody to the protein of interest. All blocking, washing, and antibody incubation steps were done using both- conventional method of submerging the membrane in a liquid solution and our new cool-mist-based system. The bound antibodies are then detected by imaging the membrane using appropriate imaging equipment. Detection of IRDye 800CW secondary antibodies by LICOR-Odyssey Infrared Imaging System is illustrated in the accompanying examples. The thickness of the band corresponds to the amount of protein present in the sample.

The conventional method of western blotting involves a series of manual steps prior to visualization; many were individually short but collectively require significant hands-on time. Efforts have been made to improve Western blotting by reducing manual steps through automation. The present invention relates to an automated method of performing blot processing in Western blot employing Ultrasonic Mist driven technology or gel/ immunohistochemistry slides processing. All steps (i.e., blocking, washing, and primary and secondary antibody incubations) of immunoblotting can be efficiently performed with this ultrasonic mist system. Four tubes containing washing solution, blocking solution, primary antibody solution, and secondary antibody solution were used to carry out blocking and probing steps via cool mist- based technology. A relay was designed to carry out various steps with different time duration: l ^st washing: 2 min, blocking: 1 hour, 2 ^nd washing: 15 min, primary antibody incubation: 4 hours, 3 ^rd washing: 15 min, secondary antibody incubation: 1 hour and final 4 ^th washing: 15 minutes. The transferred PVDF/nitrocellulose membrane was exposed to the mist of different solutions as per the relay described. The piezo ceramic discs were connected to the power supply via power driver boards and run at 2 amp and 4.7V. Membrane was then imaged using LICOR-Odyssey Infrared Imaging System/other suitable imaging systems. The solid support, such as PVDF/nitrocellulose membrane obtained after application of the antigens in the present invention may be dried and stored indefinitely at ambient temperature for an indefinite period, providing that it is maintained in a dehydrated state.

Immunohistochemistry (or ETC): IHC is a method for demonstrating the presence and location of proteins in tissue sections. Immunohistochemical staining is performed with antibodies that recognize the target protein. Since antibodies are highly specific, they bind only to the protein of interest. The antigen-antibody interaction is then detected using either chromogenic detection or fluorescent detection. 5 pm thick cryostatic sections of hippocampus of rat brain were affixed on to a (3-Aminopropyl) triethoxysilane coated slides. The tissue sample can be any sample that contains cells of interest, which can be affixed on to a solid support. Although the invention is not limited to brain cell detection, that is one suitable utility thereof. Thus, tissue specimens can include specimens of tissues that are found in all sites, including, but not limited to lung, bone, liver, esophagus, heart, breast, colon and entire gastrointestinal tract, prostate and ovarian system, brain, spleen, pancreas, and skin. Once mounted, the slides were incubated overnight at room temperature to remove any moisture that may be trapped under the sections. For immunohistochemical assays, the wash solution is usually Tris-buffered saline (TBS), Phosphate buffered saline (PBS), or a similar physiologic buffer. For IHC assays performed on a slide, the buffer is not entirely eliminated from the slide and remains there to dilute the IHC reagents and to remove any unbound antibodies/reagents from the tissue section/slide and thus reducing any background signal. IX TBS has been used as awash solution in the illustrated example of the present invention. The citrate-based buffers are used for antigen retrieval, which break the protein cross-links and thus unmask the antigenic epitopes in formalin- fixed/paraffin-embedded (FFPE) tissue sections, consequently increasing the staining intensity of antibodies. In the present invention, slides containing tissue sections were washed with IX TBS (50 mM Trizma® base, 150 mM NaCl, pH =7.5) for 5 min followed by incubation in acetone for 5 minutes. The sections were again washed with TBS for 5 minutes and then incubated in preheated antigen retrieval buffer (lOmM Sodium citrate dihydrate, 0.05% Tween-20, pH=6.0) in a water bath at 90°C for 30 minutes. Slides were allowed to cool for 20 minutes before immersing in wash buffer to prevent tissue from falling off the slide. Sections were rinsed with chilled TBS for 5 min and then incubated in TBST (TBS+0.1% Triton X-100) for 10 minutes. To eliminate the problem of non-specific staining, majority of approaches depend on the use of quite highly concentrated solution of non-immune serum (1:5 to 1:20) or 2-5% BSA. The present invention used 3% BSA in TBST (TBS+0.1% Triton X-100) as a blocking agent to prevent any non-specific antibody binding. Sections were incubated with blocking solution for 1 hour at RT by immersing the slide in a petri dish containing blocking solution and by exposing slide to the mist of blocking solution using conventional and mist-based systems, respectively. Slides were then drained for a few seconds (did not rinse) to remove blocking solution. The assay region on a slide was then incubated with an appropriate primary antibody in a suitable buffer against the protein of interest for 3-4 hours at RT, followed by secondary antibody incubation for 1-2 hours at RT with subsequent washing steps with wash solution between antibodies incubation. The present invention involves exposing the assay region to the cool mist phase of primary antibody, secondary antibody, and wash solution which would enhance the probability of antigen- antibody contact. As is well established, the primary antibody is designed to bind the antigen of interest. The secondary antibody possesses specificity for the primary antibody and is labeled (i.e., conjugated with a dye or an enzyme). In a preferred embodiment, the primary antibody is raised in rabbit against Neuron-specific Anti-b-III tubulin (Dilution: 1:1000) and secondary antibody is goat raised anti-Rabbit IgG H &L(Alexa Fluor 488) (Dilution: l:1000).Any antibodies against an antigen of interest to be identified by IHC can be used with the present invention. Following incubation, washing, and air drying; the sections were subjected to signal detection using fluorophore- and enzyme- and mediated fluorescent and chromogenic detection respectively. The present invention is further demonstrated by the following specific but non- limiting examples.

EXAMPLES

The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

Example 1

This experiment was carried out to demonstrate the binding capability of Coomassie Brilliant Blue dye with protein in cool mist phase, which is usually done in liquid phase with continuous gentle shaking. SDS-PAGE gel was run using conventional protocol with 8 wells loaded with 50pgprotein per well and two wells were loaded with protein ladder. After electrophoresis, the gel was incubated in fixing solution (50% methanol, 10% glacial acetic acid) for one hour with gentle shaking. After one hour, fixing solution was changed and gel was left for overnight in fixing solution with gentle shaking. After overnight fixation, gel was cut into two equal parts and exposed to staining solution (0.125% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid). One gel was processed using conventional method, where gel was submerged in liquid staining solution and kept on shaker for 1 hour while second gel was exposed to staining solution through cool mist-based new system without any shaking. After one hour of staining, both the gels were exposed to destaining solution (40% methanol and 10% glacial acetic acid). Gel which was stained using conventional submerged and shaking method, was destained for 90 minutes, with change in destaining solution after every 15 minutes. However, gel which was stained with new cool mist system, was destained only for 45 minutes. After destaining, both gels were imaged together using G800 Calibrated Densitometer (BioRad). Images showed that gel stained using new mist-based system have more dark and clear bands of proteins in comparison to gel which was stained using conventional shaking method (Figure 1A&B). Moreover, less time was required to destain the gel because the mist-based system did not overstained the gels.

Results: Coomassie Blue Staining of the SDS-PAGE gels carried out using the claimed device has clearly detected the proteins in complete gel and intensity of the bands was better than conventional method and required only 45 minutes of destaining in comparison to 90 minutes required by conventional method.

Example 2

This experiment was carried out to demonstrate the binding capability of Coomassie Brilliant Blue dye with protein loaded in increasing or decreasing amount in cool mist phase. SDS-PAGE gel was run using conventional protocol with 8 wells loaded with increasing amount of protein (i.e., 20, 30, 40 or 50pg) and two wells were loaded with protein ladder. After electrophoresis, the gel was incubated in fixing solution (50% methanol, 10% glacial acetic acid) for one hour with gentle shaking. After one hour, fixing solution was changed and gel was left for overnight in fixing solution with gentle shaking. After overnight fixation, gel was cut into two equal parts and exposed to staining solution (0.125% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid). One gel was processed using conventional method, where gel was submerged in liquid staining solution and kept on shaker for 1 hour while second gel was exposed to staining solution through cool mist-based new system without any shaking. After one hour of staining, both the gels were exposed to destaining solution (40% methanol and 10% glacial acetic acid). Gel which was stained using conventional submerged and shaking method, was destained for 90 minutes, with change in destaining solution after every 15 minutes. However, gel which was stained with new mist system, was destained only for 45 minutes, with change in destaining solution after every 15 minutes. After destaining, both gels were imaged together using G-800 Calibrated Densitometer (BioRad). Image analysis have shown that gel stained using new mist system demonstrate better differences in intensity of protein with increase in amount of loaded proteins (Figure 2 A&B). Moreover, less time was required to destain the gel because the developed mist-based system did not overstained the gels.

Table 1: Comparison of Integrated densitometric values (IDV) of band obtained on gels using the conventional staining system sand the developed apparatus.

Example 3

This experiment was carried out using the developed cool mist-based system automated for following steps of Immunoblotting: a) Blocking, b) Washing, c) Primary antibody incubation, d) Washing e) secondary antibody incubation, f) Washing. A conventional SDS-PAGE electrophoresis was carried out and proteins were transferred from gel to PVDF (Immobilon®- FL, Cat. IPFLOOOIO) membrane using regular wet transfer method. The membranes with transferred proteins were dried and kept in between filter papers for further use. In each of the four tubes of instrument (Figure 2-5) one of the following was pipetted a) IX TBST for washing b) 1% skimmed milk + 4% bovine serum albumin (BSA) as blocking solution, c) Rabbit raised Anti-P-III-Tubulin(Dilution: 1:5000) in IX TBST solution as primary antibody, d) Anti Rabbit (Dilution: 1:2000)IR Dye ® 800 CW labeled secondary antibody in IX TBST buffer containing 0.5% skimmed milk. A relay was designed to carry out various steps with following duration and sequence: a) 2 minute washing followed by b) 1 hour blocking; c) 15 minute washing; d) 4 hours primary antibody; e) 15 minute washing; f) 1 hour secondary antibody incubation; g) 15 minute washing. In parallel, a membrane with same amount of protein was processed with conventional protocol using same time of incubation as used above. In conventional protocol, the membrane was incubated in liquid solutions of blocking, primary antibody, secondary antibody and washing solutions and kept on shaker in same sequence and at the same time as above. All above steps were carried out at room temperature. Finally, membrane was taken and directly scanned on FICOR-Odyssey Infrared Imaging System for imaging.

Results: Imaging of the membranes have shown the presence of b-III tubulin reactive protein in both the membranes, which are below the 63 kDa and above 48 kDa bands of protein ladder. Example 4

An immunoblotting experiment was performed in which secondary antibody incubation was carried out using our new system and all other steps were carried out with conventional method as described in example 3. In parallel, one membrane was processed with complete conventional protocol as described in example 3. Briefly, both the membranes were blocked with 5% skimmed milk for 1 hour. After 15 minutes of washing with IX TBST, both membranes were incubated for 4 hours in primary antibody solution (Mouse raised Anti-P-actin Antibody; Cat. A5316 from SIGMA) prepared in IX TBST with 1:5000 dilution. After 15 minutes of washing on shaker, one-hour secondary antibody incubation was carried out. One membrane was processed with conventional shaking method and second membrane was exposed with secondary antibody using our new mist-based system with single step program. All the steps were carried out at room temperature. Imaging of both membranes was carried out using LICOR-Odyssey Infrared Imaging System.

Results: Imaging of the membranes have shown the presence of b-actin reactive protein in both the membranes, which are below the 48 kDa and above 35 kDa bands of protein ladder.

Example 5

An immunoblotting experiment was performed in which secondary antibody incubation was carried out using our new system at 4 °C and 37 °C and all other steps were carried out with conventional method as described in example 3. All three membranes were blocked with 5% skimmed milk for 1 hour. After 15 minutes of washing with IX TBST, both membranes were incubated for 4 hours in primary antibody solution (Mouse raised Anti-P-actin Antibody; Cat. A5316 from SIGMA) prepared in IX TBST with 1:5000 dilution. After 15 minutes of washing on shaker, one-hour secondary antibody incubation was carried out at either 4 °C and 37 °C using new mist-based system with single step program. For achieving different temperature, new mist- based system was kept in incubator set at either 4 °C and 37 °C. All other steps were carried out at room temperature. Imaging of both membranes was carried out using LICOR-Odyssey Infrared Imaging System. Results: Imaging of the membranes have shown the presence of b-actin reactive protein in both the membranes, which are below the 48 kDa and above 35 kDa bands of protein ladder. Band intensity was better at 4 °C.

Example 6

An experiment was performed to determine the effect of solvent like methanol on mist formation of antibody and its effect on detection of protein. An immunoblotting experiment was performed in which secondary antibody incubation was carried out using our new mist-based system in presence of 10% methanol and all other steps were carried out with conventional method as described in example 3. In parallel, one membrane was processed with complete conventional protocol as described in example 1. Briefly, both the membranes were blocked with 5% skimmed milk for 1 hour. After 15 minutes of washing with IX TBST, both membranes were incubated for 4 hours in primary antibody solution (Mouse raised Anti-P-actin Antibody; Cat. A5316 from SIGMA) prepared in IX TBST with 1:5000 dilution. After 15 minutes of washing on shaker, one-hour secondary antibody incubation was carried out. One membrane was processed with conventional shaking method and second membrane was exposed with secondary antibody using new mist-based system with single step program. All the steps were carried out at room temperature. Imaging of both membranes was carried out using LICOR-Odyssey Infrared Imaging System.

Results: Imaging of the membranes have shown the presence of b-actin reactive protein in both the membranes, which are below the 48 kDa and above 35 kDa bands of protein ladder. No effect of methanol was observed in protein detection using mist system.

Example 7

An immunoblotting experiment was performed in which primary antibody incubation was carried out using our new mist-based system and all other steps were carried out with conventional method as described in example 3. Briefly, the membrane was blocked with 5% skimmed milk for 1 hour. After 15 minutes of washing with IX TBST, the membrane was incubated for 4 hours in primary antibody solution (In Rabbit, primary antibody for b-III tubulin, Abeam, 18207) prepared in IX TBST with 1:1000 dilution. After 15 minutes of washing on shaker, one-hour secondary antibody incubation was carried out. All the steps were carried out at room temperature. Imaging of membrane was carried out using LICOR-Odyssey Infrared Imaging System.

Results: Imaging of the membrane has shown the presence of b-III tubulin reactive protein in below the 63 kDa and above 48 kDa bands of protein ladder.

Example 8

An immunoblotting experiment was performed in which blocking of membrane was carried out using our new mist-based system and all other steps were carried out with conventional method as described in example 3. Briefly, the membrane was blocked with 5% skimmed milk for 1 hour using our new mist-based system. After 15 minutes of washing with IX TBST, the membrane was incubated for 4 hours in primary antibody solution (In Rabbit, primary antibody for b-III tubulin, Abeam, 18207) prepared in IX TBST with 1:1000 dilution. After 15 minutes of washing on shaker, one-hour secondary antibody (IR Dye ® 800 CW anti Rabbit in 0.5% skimmed milk in IX TBST diluted to a ratio of 1:5000) incubation was carried out. All the steps were carried out at room temperature. Imaging of membrane was carried out using LICOR-Odyssey Infrared Imaging System.

Results: Imaging of the membrane has shown the presence of b-III tubulin reactive protein in below the 63 kDa and above 48 kDa bands of protein ladder.

Example 9

This experiment was carried to demonstrate the use of our new cool mist-based system in protein detection using Immunohistochemistry (IHC). Rat brain hippocampus sections were cut in a microtome to a thickness of approximately 5 microns and affixed on to a silane coated slides. Once mounted, the slides were incubated at room temperature for overnight. Two sets of slides containing tissue sections were washed with Tris Buffer Saline (TBS) for 5 minutes and then incubated in acetone for 5 minutes. Sections were again washed with TBS for 5 minutes. Sections were then incubated in preheated antigen retrieval buffer (lOmM Sodium Citrate, 0.05% Tween-20, pH=6.0) in a water bath at 90°C for 30 minutes. Slides were allowed to cool for 20 minutes before immersing in wash buffer to prevent tissue from falling off the slide. Sections were rinsed with chilled TBS for 5 minutes and then incubated in TBS buffer containingO.1 % Triton X-100 for 10 minutes. Sections were again rinsed with TBS for 5 minutes. After washing with TBS one set of section was processed with conventional method (incubating sections directly with antibody in liquid phase) and in second set all the incubations except washing was carried out using our new cool mist-based system. Blocking was carried out with 3% BSA in TBST (TBS+0.1% Triton X-100) for 1 hour at room temperature. Slides were then drained for few seconds (did not rinse) to remove left over blocking solution. Sections were incubated with Primary antibody developed in Rabbit against Neuron specific Anti-b-III tubulin (at>18207, Abeam) at 1:1000 dilutions in IX TBST (TBS+0.1% Triton X-100) for 3.5 hours at RT. After primary antibody incubation, 3 washings of 10 minutes each were carried out in IX TBST (TBS+0.1% Triton X-100). Sections were then incubated for 90 minutes in secondary antibody raised in Goat Anti -Rabbit IgG H & L (Alexa Fluor 488) (Catalog No. A11008) prepared at dilutions of 1:1000 in IX TBST at room temperature in the dark. Sections were washed three times with TBS for 5 minutes each in the dark and then with IX TBST (TBS+0.1% Triton X- 100) for 5 minutes and final washing was carried out with MilliQ water for 5 minutes. Sections were air dried to remove any moisture and then mounted with a drop of anti-fade mounting medium with DAPI, coverslip was placed over the sections and sealed with nail polish to prevent drying and movement under the microscope. Imaging was done using EVOS® FL Auto Imaging System.

Results: Immunohistochemistry carried out in hippocampal region of rat brain section for levels of GFP protein using conventional and new mist-based system has shown the presence of GFP proteins and nuclei.

ADVANTAGES OF THE INVENTION

> The new cool mist-based system claimed here will easily automate several steps of immunoblotting, immunohistochemistry or immunocytochemistry, without the need of robotics. Staining or labeling of biomolecules in cool mist phase prevents over-staining as observed in conventional methods of gels, membranes or slides, which results in low backgrounds, less non-specific reactivity and higher staining of target biomolecules. Low background, higher staining of targeted molecules, less non-specific reactivity, automation of all the steps of immunoblotting will increase the repeatability of results and will reduce the required time and amount of antibody required. Increased repeatability, simplification of the protocols and better detection of targeted biomolecules will push techniques like immunoblotting in diagnostic setup and help in early detection of several diseases.