WO2012027219A1

WO2012027219A1 - Pre-assembled and pre-wet multilayer substrate for western blotting

Info

Publication number: WO2012027219A1
Application number: PCT/US2011/048402
Authority: WO
Inventors: Malcolm G. Pluskal
Original assignee: Advanced Bioscience Technologies, Llc
Priority date: 2010-08-23
Filing date: 2011-08-19
Publication date: 2012-03-01

Abstract

The invention pertains to packaged, pre-assembled and pre-wet multilayer substrates for analyte transfer, particularly for use with Western Blotting techniques. The multilayer substrate comprises components of traditional Western Blotting but it is pre-assembled into a laminated unit that stays together after assembly and storage. The multilayer substrate comprises a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and a layer comprising blotting material that is pre-wet, the multilayer substrate being assembled in a laminated unit.

Description

PRE-ASSEMBLED AND PRE- WET MULTILAYER SUBSTRATE FOR

WESTERN BLOTTING

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.

61/375,963, filed on August 23, 2010, The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Conventional Western Blotting of proteins requires the use of microporous membranes (PVDF or nitrocellulose), adsorbent paper and buffers to pre-wet the components. The hydrophobic PVDF membrane requires an additional pre-wetting step in a suitable alcohol (methanol or ethanol) followed by an exchange into an aqueous buffers. This process is labor intensive and time consuming and can lead to variability in the Western Blotting transfer process. Thus, a need exists for materials and methods that can simplify the Western Blotting transfer process. SUMMARY OF THE INVENTION

The invention pertains to pre-assembled and pre-wet multilayer substrates for analyte transfer, particularly for use with Western Blotting techniques. The multilayer substrate comprises components of traditional Western Blotting but it is pre-assembled into a laminated unit that stays together after assembly and storage. The multilayer substrate comprises a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and a layer comprising blotting material that is pre-wet, the multilayer substrate is assembled into a laminated unit. Preferably, the blotting material comprises a plurality of layers depending upon the operation of the substrate, i.e., whether the multilayer substrate functions as the anode or the cathode in the operation of the Western Blotting technique. In the anode embodiment, the blotting material comprises a layer comprising an adsorptive material; and a microporous membrane layer disposed between the vapor barrier layer and the absorbent layer, the absorptive layer and the microporous layer being pre-wet. In the cathode embodiment, the blotting material comprises a layer comprising an adsorptive material that is pre-wet.

The multilayer substrate is contained within a moisture impermeable housing for storage of the substrate prior to use and retains the pre-wet nature of the substrate.

In one aspect of the invention, the vapor barrier can be eliminated, provided that the pre-wet, multilayer substrate is stored in a moisture impermeable housing prior to use to avoid buffer evaporation. In this embodiment, the pre-wet, multilayer substrate comprises blotting material that is pre-wet and assembled into a laminated unit. Preferably, the blotting material comprises a plurality of layers depending upon the operation of the substrate, i.e., whether the multilayer substrate functions as the anode or the cathode in the operation of the Western Blotting technique, hi the anode embodiment, the blotting material comprises a layer comprising an adsorptive material; and a microporous membrane layer, the adsorptive layer and the microporous layer being pre-wet. In the cathode embodiment, the blotting material comprises a layer comprising an adsorptive material that is pre-wet.

In another aspect of the invention, the multilayer substrate comprises an anode unit and a cathode unit; wherein the units individually comprise a vapor barrier layer contiguously disposed on the outermost layer of the substrate; a layer comprising an adsorptive material; and optionally, for the anode unit, a microporous membrane layer disposed between the vapor barrier layer and the adsorbent layer, the absorptive layer and optionally microporous layer being pre-wet.

In a preferred embodiment, the adsorbent material will comprise at least one layer of the adsorbent material; at least two layers of the adsorbent material; at least three layers of the adsorbent material; at least four layers of the adsorbent material; at least five layers of the adsorbent material; at least six layers of the adsorbent material. Most preferred, the adsorbent material will comprise two layers of the adsorbent material.

In another embodiment, the microporous, membrane comprises

nitrocellulose or polyvinylidene fluoride. The invention also pertains to methods of making pre-wet and pre-assembled multilayer substrates for analyte transfer, particularly for use with Western Blotting techniques. The method comprises a) pre-wetting blotting material; b) stacking a plurality of the pre- wet blotting material to form layers; c) cutting the entire stack of pre-wet blotting material to thereby produce a pre- wet, multilayer substrate in a laminated unit; and d) sealing the pre-wet, multilayer substrate in a moisture impermeable housing. In a one embodiment, the entire stack of blotting material is cut by die cutting. In other embodiments, the blotting material is cut by knife, rotary blade or high pressure water jet cutting with means for compressing the stack. Any of the variations of the multilayer substrate described here can be made by this method.

The invention further pertains to the use of the pre-wet and pre-assembled multilayer substrates for analyte transfer, particularly for use with Western Blotting techniques. The method for performing a Western Blotting technique for analyte transfer, comprises a) placing a first pre-wet, multilayer substrate for analyte transfer into an analyte transfer device at the anode location, the pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, wherein the blotting material comprises a layer of microporous membrane, and optionally comprising a vapor barrier layer contiguously disposed on the microporous membrane, the multilayer substrate being assembled into a laminated unit; b) removing the vapor barrier layer, if present; c) placing an electrophoresis gel on the multilayer substrate of step (a); d) placing a second pre-wet, multilayer substrate on top of the electrophoresis gel, the second pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, and optionally a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and the multilayer substrate being assembled into a laminated unit; and removing the vapor barrier, if present; e) connecting the cathode electrode of the transfer device to affect analyte transfer. Steps (a) through (d) are repeated for each electrophoresis gel included in the method for simultaneous analyte transfer, where blocking material is placed on top of the second substrate before step (a) is repeated.

The invention provides benefits over conventional blotting materials used in Western Blotting and can eliminate most of the labor intensive steps by providing pre-wet and pre-sized multilayer assemblies ready for immediate use in this application. The assemblies can be placed inside a suitable outer container that can be sealed to eliminate moisture loss on storage under a wide range of environmental conditions, Pre-packaged and pre-wet blotting assemblies can reduce variability in Western Blotting.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

FIG. 1 shows a cross section through an anode electrode multilayer assembly and its outer storage bag.

FIG. 2 shows a cross section through a cathode electrode multilayer assembly and its outer storage bag.

FIG. 3 shows Western blotting performance after anode and cathode assemblies of the invention were stored at room temperature (22°C) for two weeks (Control).

FIG. 4 shows Western blotting performance after accelerated aging; anode and cathode assemblies of the invention were stored at elevated temperature (37°C) and at < 20% relative humidity for two weeks (Test),

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Definitions

The term "laminated" as used herein is intended to mean multilayers of blotting material that have been combined to be handled as a single layer or unit. The term "delaminated" as used herein is intended to mean multilayers of blotting material that have been combined to be handled as a single layer or unit but the single layer or unit partially or completely separates into its original component layers.

The term "moisture impermeable" as used herein is intended to mean material able to prevent transfer of liquid across its structure thus able to retain liquid on one side of the material.

The term "vapor barrier" as used herein is intended to mean any material, typically a plastic or foil sheet that resists diffusion of moisture through its structure.

The term "pre-wet" as used herein is intended to mean structure of the material that is fully hydrated with an aqueous liquid in advance if its use in the intended application.

The term "pre-assembled" as used herein is intended to mean material that has been cut to desired size, hydrated with an aqueous buffer and assembled with other components into a final assembly ready to use in the blotting application.

The term "assembly" as used herein is intended to mean the pre-assembled and pre-wet multilayer substrates of the invention, either separately as the anode and cathode portions or together with the electrophoresis gel.

The term "analyte" as used herein is intended to mean a compound or subject to be detected. Preferred anaiytes include, but are not limited to, polypeptides and nucleic acids and detectable fragments thereof.

The term "analyte transfer device" as used herein is intended to mean any transfer device that can be used for Western Blotting. Transfer devices are intended to include devices based on a semi-dry format with flat plate electrodes, and devices based on a buffer tank with flat plate or wire electrodes.

The term "blotting material" as used herein is intended to mean any material that is used in the art of blotting, including but not limited to, the adsorbent layer and the microporous layer.

The term "blocking material" as used herein is intended to mean any material that acts as a barrier to prevent cross talk of anaiytes between multiple assemblies. The proteins are blocked but not the ions necessary for conductivity during the blotting process. Examples of suitable materials are dialysis membranes as described herein.

Singular and plural usage of these definitions is contemplated herein, The invention pertains to pre-assembled and pre- wet multilayer substrates for analyte transfer, particularly for use with Western Blotting techniques. As used herein the term "multilayer substrate" is intended to mean the pre-assembled and pre-wet layers of blotting material and optionally vapor barrier. The multilayer substrate comprises components of traditional Western Blotting but it is pre- assembled into a unit that stays together after assembly and storage. The unit appears to be "laminated" or compressed together by the process of cutting the stacked multilayer substrate, such that under handling and storage conditions, the multilayer substrate does not delaminate or fall apart. The laminated unit therefore conveniently facilitates the assembly of the parts needed to perform analyte transfer, such as by Western Blotting. Because the layers of the multilayer substrate stay together, the need to remove bubbles between layers in traditional assembly is eliminated. The multilayer substrates of the invention eliminate many of the manual steps in traditional Western Blotting techniques, but traditional buffers, transfer devices, techniques and microporous membranes can all be used with the substrates of the invention. Thus, the invention provides a convenient tool in Western Blotting that is readily useable in Western Blotting formats and protocols.

In addition to traditional components for Western Blotting, there is provided a vapor barrier or impermeable cover sheet on the outermost layer of the assembly to prevent the assembly from drying out during handling of the assembly. The vapor barrier or cover sheet allows the assembly to be manipulated while retaining the pre- wetted nature of the blotting material. Further, the vapor barrier can include a visual indicator, for example the vapor barrier can be color coded to identify the assembly as the anode or the cathode. It is preferred to color code the anode blue/black and the cathode red, or other color used by the skilled person to identify the cathode and anode. In addition to providing a vapor barrier, the cover sheet can be marked or printed with information on use, electrode and orientation to the electrode, buffer chemistry and membrane type, or other instructional information to aid in use of the assembly. The vapor barrier can have a tab or overhang that can facilitate removal of the vapor barrier during use.

The vapor barrier/coversheet can be a non-porous polyethylene film that can optionally be colored and printed for certain embodiments of the invention, as described herein. Commercially available materials that may be suitable vapor barriers include, for example, ACLAR, a polychlorotrifluoroethylene (PCTFE) made by Allied-Signal or other polymers, such as Saran (poly vinylidene chloride) or Tedlar (polyvinyl fluoride, Du Pont). Preferably, the vapor barrier/coversheet is a material that will not impart UV leachables to the assembly, particularly under storage conditions, and therefore will not increase the fluorescence background of the assembly, such as a non-UV leaching polyethylene film. Trace amounts of UV leachables may be acceptable provided that they do not increase the fluorescence background to a level that will interfere with the desired fluorescent signal from the analyte.

Since the multilayer substrate is assembled as a unit that is pre-wet and the pre-wet character needs to be preserved, the multilayer substrate should be packaged in a moisture impermeable housing, such as a storage bag. The housing will protect the multilayer substrate from drying out and delaminating. In embodiments that utilize a vapor barrier, when the assembly is removed from the outer storage bag, the vapor layer provides: a) a vapor barrier to reduce moisture loss from the membrane layer by evaporation; b) a protective layer to keep the membrane free of

contamination during handling after opening the package; and c) a visual indication of the anode or cathode components and the location of the microporous membrane in the multilayer product. These assemblies within their outer storage container will have an enhanced shelf life over a wide range of environmental conditions.

The moisture impermeable housing can be a thin metal foil (Aluminum), Mylar film, polyethylene coated with aluminum, or non-porous polyethylene film. Suitable products that can be used for the housing are FOIL-O-RAP Fr2176 (aluminized) by Bell Fibre Products, Columbus, GA, and MARVELS EAL 360 (aluminized) by Ludlow Corporation, Laminating and Coating Divisions, Homer, LA. During the manufacturing process the pre- wet multilayer substrate is cut in one unit operation to yield an assembly that handles like a single stable component. In addition, the individual layers will be in uniform contact and free of any air bubbles that can be introduced during manual assembly. This process enhances the handling of these multilayer components when assembling the Western Blotting devices. According to one embodiment, the method comprises a) pre- wetting blotting material; b) stacking a plurality of the pre- wet blotting material to form layers; c) cutting the entire stack of pre-wet blotting material to thereby produce a pre-wet, multilayer substrate in a laminated unit; and d) sealing the pre-wet, multilayer substrate in a moisture impermeable housing. In a one embodiment, the entire stack of blotting material is cut by die cutting. In other embodiments, other cutting techniques can be employed such as knife, rotary type blade or high pressure water jet cutting with a means for compressing the layers during cutting. Any of the variations of the multilayer substrate described here can be made by this method. The resulting multilayer substrate will be cut to desirable size, for example, they can be cut into sizes compatible with commercially available pre-cast SDS-PAGE gels, e.g., 8x8 cm (for mini gels) and 8x13 cm (for Biorad Criterion gels). Any method of pre-wetting the blotting material can be used in the assemblies and methods described herein. The skilled person would know the materials used for pre-wetting the membranes based upon the type of membrane used in the assembly.

In one embodiment, pre-wetting of a PVDF membrane comprises contacting the membrane with methanol or ethanol followed by an exchange in water. For assemblies comprising nitrocellulose, alcohol pre-wetting in not required. After the membrane is pre-wet, the adsorbent material and the membrane are contacted (e.g., soaked) in an excess of liquid to saturate the structure of the adsorbent and membrane.

The anode unit assembly comprises a microporous membrane, whereas the cathode unit assembly does not comprise a microporous membrane. Any microporous membrane that is used in Western Blotting is contemplated for use in the subject invention. Microporous membranes that are suitable for use in the subject invention include but are not limited to nitrocellulose, polyvinylidene fluoride (PVDF), polyethylene, polypropylene, polyethersulphone, polytetrafluoroethylene. Preferably, the microporous membrane will be

nitrocellulose or PVDF, Any of these membranes can be surface modified to offer retention by ion exchange, reverse phase, metal ion chelation and specific ligand affinity. Contemplated examples include Protein A or equivalent,

Steptavidin/avidin, lectin or biomimetic ligands based on peptides.

In one embodiment, a hydrophilic blotting membrane described in

US20100044302 Al "Hydrophilic, high protein binding, low fluorescence, Western Blotting membrane" can be used. The hydrophilic membrane can be used in Western Blotting and eliminates the alcohol pre-wetting required with PVDF. In another embodiment, blotting material can be those described in U.S. Patent Number 5,004,543, entitled "Charged-Modified Hydrophobic Membrane Materials and Method for Making the Same." The entire teachings of these references are incorporated herein by reference.

Any adsorbent material that is traditionally used in Western Blotting can be used in the invention. For example, Whatman chromatographic paper (e.g., 3 MM), adsorbent paper having high cotton lint content, or the equivalent can be used. In a preferred embodiment, the adsorbent material will comprise at least one layer of the adsorbent material; at least two layers of the adsorbent material; at least three layers of the adsorbent material; at least four layers of the adsorbent material; at least five layers of the adsorbent material; at least six layers of the adsorbent material. In a most preferred embodiment, the adsorbent material will comprise two layers of the adsorbent material. The layers of adsorbent material can be the same material used for each layer or they can be a plurality of different layers of adsorbent material.

The invention is intended to cover the use of at least one multilayer substrate in the assembly of materials for accomplishing analyte transfer. For example, the user can use the anode portion of the multilayer substrate of the invention, place the gel thereon and then apply blotting material (either the pre-assembled substrate of the invention or traditional layering of the adsorbent) onto the gel to complete the assembly. Any combination of use of the multilayer substrates of the invention, with or without traditional Western Blotting techniques and materials, are contemplated. The individual multilayer components will be manufactured pre-wet in the appropriate transfer buffer for Western Blotting and cut as a single unit to yield a multilayer assembly that is ready for use in a Western Blotting technique or can be packaged for storage until it is ready for use. In FIG. 1 , the multilayer Anode electrode assembly 5 comprises an adsorbent layer 1 comprising sheets of Whatman (e.g., 3 MM) chromatography paper or equivalent, a single microporous membrane 2 comprising PVDF, nitrocellulose or equivalent substrate used for Western Blotting and a cover sheet layer 3 comprising of a suitable colored or printable non-porous material, such as polyethylene film or equivalent acting as a vapor barrier and visual indicator. The assembly 5 is then sealed into an outer bag 4 constructed of a suitable moisture impermeable material, such as metal ized-polyethylene multilayer or equivalent able to maintain a constant humidity level for stable storage under a wide range (e.g., 4-55°C and 0-75% relative humidity) of environmental conditions. In FIG. 2, the multilayer Cathode electrode assembly 6 consists of an adsorbent layer 1 comprising sheets of Whatman 3MM chromatography paper or equivalent and a cover sheet layer 3 comprising of a suitable colored or printable non-porous material, such as polyethylene film or equivalent acting as a vapor barrier and optionally a visual indicator. The assembly 6 is then sealed into an outer bag 4 constructed of a suitable moisture impermeable material, such as a metalized- polyethylene multilayer or equivalent able to maintain a constant humidity level for stable storage under a wide range (e.g., 4-55°C and 0-75% relative humidity) of environmental conditions. It should be understood that the multilayer substrates do not require the need for electrodes in the assembly; reference is made to anode or electrode for the convenience of the location where the substrate will be placed in the transfer device.

The invention further pertains to kits for using the multilayer substrates of the invention. One or more multilayer substrates can be packaged together or individually. For example, the anode portion of the multilayer substrate can be packaged together, or individually, with the cathode portion of the multilayer substrate. Alternatively, a plurality of same multilayer substrates can be packaged together, e.g., a plurality of the anode portion of the multilayer substrate can be packaged together for use in Western Blotting a plurality of gels at the same time. In this example, multiple anode portions will be needed but only one cathode portion will be used on the assembled stack of alternating anode multilayer substrate and gel layers. The components of the kit are packaged in a suitable container for shipping. Additional reagents can be optionally included, for example, reagents for detecting transferred analytes, written instructions for using the membranes and buffers for use with the membranes.

For example, in one embodiment the kit, comprises an anode and cathode assembly set, each being separately packaged in a moisture impermeant housing; the Anode portion of the assembly set comprises a pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, wherein the blotting material comprises a layer of microporous membrane, and optionally comprising a vapor barrier layer contiguously disposed on the microporous membrane, the multilayer substrate being assembled into a laminated unit; the Cathode portion of the assembly set comprises a pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, and optionally a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and the multilayer substrate being assembled into a laminated unit; and instructional material.

OPERATION

In operation, the above multilayer assemblies will be used in conventional

Western Blotting applications fully compatible with all commercially available semi-dry and tank device formats. They will be pre-wet with buffers, widely described in the literature and will not require any proprietary buffer formulation. The outer container will provide storage conditions for enhanced shelf life under a wide range of storage conditions. In operation the multilayer assemblies will be used in Western Blotting as follows:

1. Open the Anode multilayer assembly outer package, remove and place on the Anode electrode (semi-dry) or the anode side of the tank blotting assembly, with the cover sheet on top. Note: the cover sheet prevents the membrane surface from drying out and allows extra time for assembly of the Western Blotting transfer stack.

2. Prepare the electrophoresis gel for Western Blotting transfer, 3. Remove the Anode cover sheet and place the gel on the membrane surface.

4. Open the Cathode multilayer assembly outer package, locate

colored/marked side of the assembly - remove the cover sheet to expose a clean surface which is then place on top of the gel to complete the blotting assembly.

5. Place the Cathode electrode in place (semi-dry) or close the tank blotting assembly making sure the anode assembly is orientated facing the Anode electrode in the tank transfer device. in the above process, the manual handling of the components involved in Western Blotting will be reduced to three steps including application of the electrophoresis gel. No additional buffer is required for semi-dry transfer.

The above operation discussion illustrates the use of a single electrophoresis gel, however the invention is not limited to Western Blotting with only a single gel. For blotting multiple gels, the pre- wet, multilayer substrates can be stacked as any traditional Western Blotting assembly is set up. For example, the anode multilayer assembly of the invention is placed on the anode electrode and, if present, the cover sheet is removed. The electrophoresis gel is placed on the anode assembly and then the cathode assembly of the invention is placed thereon. If a coversheet or vapor barrier is present on the cathode assembly, it will be removed prior to placement on the gel. Because several gels will be blotted simultaneously, it is necessary to place a dialysis membrane or the equivalent, between each anode/gel/cathode assembly to prevent cross talk between the blotting. The process can be repeated one time, two times, three times, four times, five times for up to six gels. Examples of dialysis membranes suitable for use in the invention include but are not limited to regenerated cellulose semi-permeable membrane or regenerated cellulose film known as Cellophane

In certain embodiments, Western Blotting can be performed using techniques described in US20100032296 Al describes "Systems and methods for quantitative analyte transfer". The focus of the methodology described in this application is enhancement of retaining analytes of lower molecular weight, termed "blow- through" by the adsorptive membrane employed in Western blotting. The entire teachings of this reference are incorporated herein by reference.

The following examples are provided by way of illustration only and are not intended in any way to be limiting. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

EXAMPLE 1 - Effects of vapor barrier materials on moisture loss rate from pre- wet, multilayer substrate

A series of 8cm x 8cm pre-wet, multilayer substrate assemblies (2 layers of paper + 1 layer of PVDF blotting membrane) were prepared from components as described above and provided with a range of vapor barrier materials to test the moisture loss rate due to evaporation. This was measured by placing the assembly on a sheet of non-porous aluminum foil and measuring the total weight after a range of time intervals up to 60 min. The moisture loss rate was expressed as volume loss/minute per unit area of the assembly at 5, 30 and 60 min time points. The results are summarized in Table 1 below.

TABLE 1

Moisture loss rate from pre-wet multilayer substrate

* Metal side down in contact with PVDF membrane

The above results show a clear reduction (> 75%) in moisture loss rate from the surface of the pre-wet multilayer assemblies when compared to NO vapor barrier. The non-porous polyethylene sheet was the preferred material showing the highest moisture loss rate reduction. EXAMPLE 2 - Accelerated shelf life testing of packaged pre-wet, multi-layer assemblies

Accelerated shelf life testing at elevated temperatures is routinely used to assess the stability of products in shorter time periods. The data can then be extrapolated to longer shelf life estimates at sub-ambient or room temperature. This type of study was carried out to test the stability of pre-wet, multi-layer assemblies sealed in a moisture impermeable housing for storage at elevated temperatures. This approach has been applied to food, pharmaceuticals and cosmetics to more rapidly estimate the product stability at ambient (from about 20 to about 25°C) to sub- ambient temperatures (about 4°C).

Samples of packaged pre-wet, multi-layer assemblies (eBlot PVDF Cat# EBVFSDAX2, Advanced Bioscience Technologies LLC) that had been vacuum sealed in metalized laminated film bags (Laminated Films & Packaging,

Portsmouth, NH) were placed into an environmental chamber maintained at 37°C / <20% relative humidity for 2 weeks. Control packaged samples were stored at room temperature (22°C) in parallel for the same time period. Western blot testing was carried out using: a) Whole human serum sample (2 mg/mL total protein) prepared in IX SDS sample buffer (2.0% [Wt./v] SDS, 0.112M Tris Acetate buffer pH 7.0 and 10 mM Dithiothreitol) were heated to 95 °C for 3 min. After cooling to room temperature 10 μΤ was loaded per well in lanes 2-9 and b) colored molecular weight markers (5 μΕ Precision Plus Kaleidoscope Protein Standards, Cat# 161-0375 from Bio-Rad, Hercules, CA) in lanes 1 and 10 of a pre-cast 12% SDS-PAGE gels (TGX Cat#456-1043S from Bio-Rad, Hercules, CA) in Tris-Glycine running buffer. The gels were run at 200 v for 45 min or until the blue dye reached the bottom 1 cm of the gel cassette. After removing the gel from the cassette, the Western blotting transfer was carried out as described in OPERATION at 24 v for 12 min in a semi- dry blotting apparatus (eBlotter Cat# EBSD1010 from Advanced Bioscience Technologies, LLC). The resulting membranes were visualized with a reversible stain (MemCode™ from Thermo-Fisher Pierce Technology Products, Rockford, IL) and destained in water and then air dried. The dried membranes were then scanned using an Epson 3200 Photo Scanner and stored as 8-bit grey scale images. The results of the transfer of colored markers and whole human plasma from this accelerated aging study are summarized in FIG. 3 and FIG, 4. FIG. 3 shows Western blotting performance after anode and cathode assemblies of the invention were stored at room temperature (22°C) for two weeks (Control). FIG. 4 shows Western blotting performance after accelerated aging; anode and cathode assemblies of the invention were stored at elevated temperature (37°C) and at < 20% relative humidity for two weeks (Test).

The eBlot packaged pre- wet, multi-layer assemblies showed < 0.1% loss of weight over the two week study at 37°C and the resulting Western blot transfers showed no significant differences, indicating full functional performance of the product after accelerated ageing at 37°C for 2 weeks.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A pre-wet, multilayer substrate for analyte transfer, comprising:

a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and a layer comprising blotting material that is pre-wet, the multilayer substrate being assembled in a laminated unit.

2. The multilayer substrate of Claim 1, wherein the vapor barrier includes a visual indicator.

3. The multilayer substrate of Claim 1 , wherein the vapor barrier further

comprises instructional information.

4. The multilayer substrate of Claim 1, wherein the blotting material comprises a layer of adsorbent material.

5. The multilayer substrate of Claim 4, wherein the adsorbent material

comprises at least one layer of the adsorbent material; at least two layers of the adsorbent material; at least three layers of the adsorbent material; at least four layers of the adsorbent material; at least five layers of the adsorbent material; at least six layers of the adsorbent material.

6. The multilayer substrate of Claim 1 , wherein the blotting material comprises a layer of microporous membrane.

7. The multilayer substrate of Claim 6, wherein the microporous membrane is nitrocellulose or polyvinylidene fluoride (PVDF),

8. The multilayer substrate of Claim 1 , further comprising a vapor impermeable housing to store the multilayer substrate therein.

9. The multilayer substrate of Claim 1 , wherein the housing comprises

metalized polyethylene.

A pre-wet, multilayer substrate for analyte transfer, comprising:

a vapor barrier layer contiguously disposed on the outermost layer of the substrate;

a plurality of adsorbent layers assembled in a stack and pre-wet; optionally, a microporous membrane layed disposed between the vapor barrier layer and the absorbent layers;

wherein the multilayer substrate being assembled in a laminated unit; and

a moisture impermeable housing for storage of the substrate therein.

A pre-wet, multilayer substrate for analyte transfer, comprising;

blotting material that is pre-wet and being assembled in a laminated unit.

A method of forming a pre-wet, multilayer substrate for analyte transfer, comprising:

a) pre- wetting blotting material;

b) stacking a plurality of the pre-wet blotting material to form layers; c) cutting the entire stack of pre-wet blotting material to thereby produce a pre-wet, multilayer substrate in a laminated unit; and

d) sealing the pre-wet, multilayer substrate in a moisture impermeable housing.

The method of Claim 12, wherein the pre-wet blotting material comprises a layer of adsorbent material.

The method of Claim 13, wherein the adsorbent material comprises at least one layer of the adsorbent material; at least two layers of the adsorbent material; at least three layers of the adsorbent material; at least four layers of the adsorbent material; at least five layers of the adsorbent material; at least six layers of the adsorbent material.

15. The method of Claim 12, wherein the blotting material comprises a layer of microporous membrane. The method of Claim 15, wherein the microporous membrane is

nitrocellulose or polyvinylidene fluoride (PVDF).

The method of Claim 12, wherein the pre- wet, multilayer substrate comprises

a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and a layer comprising blotting material that is pre- wet.

The method of Claim 17, further comprising a layer of microporous membrane disposed between the layer of blotting material and the vapor barrier.

A Western Blotting method for analyte transfer, comprising: a) placing a first pre-wet, multilayer substrate for analyte transfer into an analyte transfer device at the anode location, the pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, wherein the blotting material comprises a layer of microporous membrane, and optionally comprising a vapor barrier layer contiguously disposed on the microporous membrane, the multilayer substrate being assembled into a laminated unit;

b) removing the vapor barrier layer, if present;

c) placing an electrophoresis gel on the multilayer substrate of step (a); d) placing a second pre-wet, multilayer substrate on top of the electrophoresis gel, the second pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, and optionally a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and the multilayer substrate being assembled into a laminated unit; and removing the vapor barrier if present; and

e) connecting the cathode electrode of the transfer device to affect analyte transfer.

The method of Claim 19, wherein the vapor barrier includes a visual indicator. The method of Claim 19, wherein the vapor barrier further comprises instructional information.

The method of Claim 19, wherein the blotting material comprises a layer of adsorbent material.

The method of Claim 22, wherein the adsorbent material comprises at least one layer of the adsorbent material; at least two layers of the adsorbent material; at least three layers of the adsorbent material; at least four layers of the adsorbent material; at least five layers of the adsorbent material; at least six layers of the adsorbent material.

The method of Claim 19, wherein the microporous membrane is

nitrocellulose or polyvinyl idene fluoride (PVDF),

The method of Claim 19, wherein steps (a) through (d) are repeated for each electrophoresis gel included in the method for simultaneous analyte transfer, where blocking material is placed on top of the second substrate before step (a) is repeated.

A kit, comprising:

an anode and cathode assembly set, each being separately packaged in a moisture impermeant housing;

the anode comprises a pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, wherein the blotting material comprises a layer of microporous membrane, and optionally comprising a vapor barrier layer contiguously disposed on the microporous membrane, the multilayer substrate being assembled into a laminated unit; the cathode comprises a pre-wet, multilayer substrate comprising a layer comprising blotting material that is pre-wet, and optionally a vapor barrier layer contiguously disposed on the outermost layer of the substrate; and the multilayer substrate being assembled into a laminated unit;

and instructional material.