Protein transfer is a vital step in western blot analysis which involves the transfer of proteins separated in a gel by electrophoresis to a solid support matrix. Immobilizing the protein to a solid support matrix facilitates the detection of specific proteins using antibodies directed against the protein(s) of interest. Typical solid matrices are membrane sheets of nitrocellulose, PVDF, or nylon. This article reviews and compares transfer methods, addresses the properties of membranes and why to choose one over another, and provides recipes for the various transfer buffers used in western blot transfer.
Western blotting of proteins was introduced by Towbin et al. in 1979 and is now a routine and fundamental technique for protein analysis. Western blotting, also called protein blotting or immunoblotting, uses antibodies to identify specific protein targets bound to a membrane; the specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture, such as cell or tissue lysate. Western blotting can be used to generate qualitative and semi-quantitative data regarding a protein of interest.
The first step in a western blotting procedure is to separate the proteins in a sample by size using denaturing gel electrophoresis (i.e., sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS-PAGE) or native PAGE. After electrophoresis, the separated proteins are transferred, or "blotted", onto a solid support matrix, usually a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. In procedures where protein separation is not required, the sample may be directly applied to the membrane by spotting using an approach called dot blotting.
Protein transfer from gel to membrane is necessary for two reasons:
- Better handling capability offered by the membrane compared to a fragile gel
- Better target protein accessibility on the membrane by macromolecules like antibodies
After transfer, the membrane must be blocked to prevent non-specific binding of the antibody to the membrane surface. The transferred protein is then probed sequentially with antibodies and detection probe (e.g., enzyme, fluorophore, isotope). An appropriate method is then used to detect the localized probe to document the location and relative abundance of the target protein.
In addition to the challenges of immunodetection in the protein blotting workflow, the transfer of proteins from a gel matrix to a membrane is a potential hurdle. The efficiency of protein transfer can be affected by the chemistry, thickness of the gel, the molecular weight of the proteins being transferred, the type of membrane and transfer buffers used, and the transfer method.
There are a variety of methods for transfer, including diffusion transfer, capillary transfer, heat-accelerated convectional transfer, vacuum blotting, and electroblotting (electrotransfer). Among these methods, electroblotting has emerged as the most popular and highly used for western blotting because it is faster and more efficient than the other methods. There are three ways to electrotransfer proteins from SDS-PAGE or native gels to membranes:
Electroblotting or electrotransfer methods rely on the electrophoretic mobility of proteins to move them out of a gel. The techniques involve placing a protein-containing polyacrylamide gel in direct contact with a piece of nitrocellulose membrane, PVDF membrane, or other suitable protein-binding support. Next, the gel-membrane pair is “sandwiched” between two electrodes, which are typically submerged in a conducting solution (transfer buffer). When an electric field is applied, the proteins move out of the gel and onto the surface of the membrane, where the proteins become tightly attached. The resulting membrane is a copy of the protein pattern that was in the polyacrylamide gel.
Schematic of western blot transfer of proteins from a polyacrylamide gel to a membrane.
Wet or Tank Transfer
When performing a wet transfer, the gel is first equilibrated in transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and the tank is filled with transfer buffer.
Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. A high field option exists for transferring a single gel, which may bring transfer time down to as little as 30 minutes, but it requires the use of high voltage (up to 200 V) or high current (up to 1.6 A) and a cooling system to dissipate the tremendous heat produced.
Transfer efficiencies of 80–100% are achievable for proteins between 14–116 kDa. The transfer efficiency improves with increased transfer time and is better, in general, for lower molecular weight proteins than higher molecular weight proteins. With increasing time, however, there is a risk of over-transfer (stripping, blow through) of the proteins through the membrane, especially for lower molecular weight (<30 kDa) proteins when using membranes with a larger pore size (0.45 µm).
Schematic showing the assembly of a typical tank transfer western blot apparatus with the position of the position of the gel, transfer membrane, and direction of protein in relation to the electrode position.
Semi-dry electroblotting (Semi-dry transfer)
In a semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes. Transfer speed is improved over wet tank by maximizing the current passing through the gel instead of around the gel. To do this, the amount of buffer used in the transfer is limited to what is contained in the transfer sandwich. In this technique it is critical that the membrane and filter paper sheets are cut to the gel size without overhangs and the gel and filter paper are thoroughly equilibrated in transfer buffer. The use of extra-thick filter paper is commonly used (approximately 3 mm thickness) to hold more transfer buffer for transfer.
Methanol may be included in the transfer buffer, but typically omitted. Electrotransfer is performed either at constant current (0.1 up to ~0.4 A) or voltage (10 to 25 V) for 10 to 60 minutes. Fast-blotting techniques use higher ionic strength transfer buffers without methanol and a high current power supply to decrease transfer times less than 10 minutes. In rapid methods, amperage is held constant and voltage is limited to a maximum of 25 V.
Semi-dry electroblotting transfer. The Invitrogen Power Blotter is designed specifically for rapid semi-dry transfer of 10–300 kDa proteins from polyacrylamide gels to nitrocellulose or PVDF membranes in 5 to 10 minutes. The Power Blotter features an integrated power supply optimized to enable consistent, high-efficiency protein transfer when used with commonly used precast or homemade gels (SDS-PAGE) and nitrocellulose or PVDF membranes.
Dry electroblotting (Dry transfer)
Dry electroblotting methods use a specialized transfer sandwich containing innovative components that eliminate use of traditional transfer buffers. A unique gel matrix (transfer stack) that incorporates buffer is used instead of buffer tanks or soaked filter papers. The high ionic density in the gel matrix enables rapid protein transfer. During blotting, the copper anode does not generate oxygen gas as a result of water electrolysis, reducing blot distortion. Conventional protein transfer techniques, including wet and semi-dry, use inert electrodes that generate oxygen. Typically, transfer time is reduced by the shortened distance between electrodes, high field strength and high current. As buffers do not need to be prepared, setup and cleanup times are greatly shorted compared to the other transfer methods.
Dry electroblotting transfer. The Invitrogen iBlot 2 Dry Blotting System provides fast western transfer without the need for buffers. This system efficiently blots proteins from acrylamide gels in 7 minutes or less, and is compatible with both PVDF and nitrocellulose membranes. The iBlot 2 System has performance comparable to traditional wet transfer methods in a fraction of the time.
Comparison of western blot transfer methods: wet, semi-dry and dry transfer methods
Efficient and reliable protein transfer from the gel to the blotting membrane is the cornerstone of a successful western detection experiment. Accuracy of results is dependent on the transfer efficiency of the western blotting method. Traditional wet transfer offers high efficiency, but at a cost of time and hands-on effort. Semi-dry blotting provides more convenience and time savings compared to traditional wet transfer, with flexibility to use multiple types of buffer systems or pre-assembled or build-it-yourself transfer stacks. However, semi-dry transfer can have a lower efficiency of transfer of large molecular weight proteins (>300 kDa). Dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.
|Semi-dry transfer||Dry transfer|
|Transfer time||30–120 min||7–10 min||5–7 min|
|Transfer buffer requirements||Requires methanol (~1000 mL)||Methanol-free transfer buffers (~200 mL)||No buffer required|
|Performance (transfer efficiency)||+++||++||+++|
|Ease of use||++||+++||+++|
|Cleanup||Extensive clean-up after each use including hazardous methanol waste disposal||Light clean-up required after each use||Very minimal with extended use|
|Special considerations||Cooling may be required for longer transfers||Multiple methods can be used including Towbin buffers||Requires pre-assembled transfer stacks|
Other methods of transfer
Diffusion blotting relies on the thermal motion of molecules, which causes them to move from an area of high concentration to an area of low concentration. In blotting methods, the transfer of molecules is dependent upon the diffusion of proteins out of a gel matrix and absorption to the transfer membrane. As the absorbed proteins are "removed" from solution, it helps maintain the concentration gradient that drives proteins towards the membrane. Originally developed for transferring proteins from (isoelectric focusing) IEF gels, diffusion blotting is also useful for other macromolecules, especially nucleic acids. Diffusion blotting is most useful when preparing multiple immunoblots from a single gel. Blots obtained by this method can also be used to identify proteins by mass spectrometry and analyze proteins by zymography. Protein recoveries are typically 25–50% of the total transferrable protein, which is lower than other transfer methods. Additionally, protein transfer is not quantitative. Diffusion blotting may be difficult for very large proteins in SDS-PAGE gels, but smaller proteins are typically easily transferred.
Vacuum blotting (Vacuum capillary blotting)
Vacuum blotting is a variant of capillary blotting, where buffer from a reservoir is drawn through a gel and blotting membrane into dry tissue paper or other absorbent material. Vacuum blotting uses a slab gel dryer system or other suitable gel drying equipment to draw polypeptides from a gel to membrane, such as nitrocellulose. Strong pumps cannot be used because the high vacuum will shatter the gel or transfer membrane. Gels may dry out after 45 minutes under vacuum, requiring plenty of reserve buffer. Gels also have a tendency to adhere to the membrane after transfer, but rehydration of the gel can help facilitate separation.
The transfer efficiency of vacuum blotting varies within a range of 30 to 65%, with low molecular weight proteins (14.3 kDa) at the high end of this efficiency range and high molecular weight proteins (200 kDa) at the low end. Like diffusion blotting, vacuum blotting allows only a qualitative transfer.
Western blot transfer conditions
Depending on the transfer method used, the voltage and time used for transfer will vary. Below are example conditions used with the different transfer methods. Depending on the molecular weight of the target protein, transfer times can be decreased (small molecular weight proteins) or increased (high molecular weight proteins) for more efficient transfers.
Western blot transfer voltage and times
|Method||Condition held constant||Time|
|Wet tank transfers|
|Mini Gel Tank||Nitrocellulose: 10 V||60 min|
|PVDF: 20 V||60 min|
|XCell SureLock Mini Cell||Tris-Glycine & Tricine: 25 V||1–2 hr|
|Bis-Tris & Tris-Acetate: 30 V||60 min|
|SureLock Tandem Midi Gel Tank||All gels and membranes: 25 V||30 min|
|Preassembled transfer stacks (with incorporated cathode and anode buffers)||1.3 Amps||5–12 min|
|High ionic buffer (1-Step Transfer buffer)||1.3 Amps||7–12 min|
|Towbin Buffer (standard semi-dry transfer)||25 V||60 min|
|iBlot Transfer Device||20–25 V||7–10 min|
The most common immobilization membranes for western blotting are nitrocellulose, PVDF, and nylon. These membranes are commonly used because they offer:
- Large surface area-to-volume area ratio
- High binding capacity
- Extended storage of immobilized macromolecules
- Ease of use
- The potential to be optimized for low background signal and reproducibility
Western blot membranes are typically supplied in either sheets or rolls, and commonly have a thickness of 100 µm, with typical pore sizes of 0.1, 0.2 or 0.45 µm. Most proteins can be successfully blotted using a 0.45 µm pore size membrane, while a 0.1 or 0.2 µm pore size membrane is recommended for low molecular weight proteins or peptides (<20 kDa).
Nitrocellulose membranes are a popular matrix used in protein blotting because of their high protein-binding affinity, compatibility with a variety of detection methods, and the ability to immobilize proteins and glycoproteins. Nitrocellulose membranes may also be used for the following applications: southern and northern blots, amino acid analysis, and dot/slot blot. Nitrocellulose membranes have a protein binding capacity of 80–100 µg/cm2. Protein immobilization is thought to occur by hydrophobic interactions, and high salt and low methanol concentrations improve protein immobilization to the membrane during electrophoretic transfer, especially for proteins with higher molecular weights. Nitrocellulose membranes remain a popular choice due to the high efficiency of irreversible protein binding.
PVDF membranes have a high binding affinity for proteins and nucleic acids and may be used for applications such as western, southern, northern and dot blots. PVDF membranes are highly hydrophobic and must be pre-wetted with methanol or ethanol prior to submersion in transfer buffer. In these applications, binding likely occurs via dipole and hydrophobic interactions. PVDF membranes have a protein binding capacity of 170–200 µg/cm2 and offer better retention of adsorbed proteins than other supports because of the greater hydrophobicity. Due to the hydrophobicity of PVDF membranes, these are the preferred choice for hydrophobic proteins (i.e., membrane proteins). PVDF is less brittle and fragile than nitrocellulose and may be useful for western blotting experiments requiring multiple rounds of reprocessing (stripping and reprobing procedures) for different targets using a new combination of antibodies.
Charged nylon (polyamide) membranes bind proteins and nucleic acids by ionic, electrostatic, and hydrophobic interactions. Nylon membranes are highly sensitive, provide consistent transfer results, and have a protein binding capacity of 480 µg/cm2. The high durability of nylon membranes offers advantages in western blotting experiments requiring stripping and reprobing procedures. A significant drawback to using nylon membranes for blotting applications is the possibility of non-specific binding and strong binding to anions like SDS.
Comparison of blotting membranes
When choosing a membrane, a protein's properties (i.e., charge, hydrophobicity) and the downstream application will determine which membrane to use. Finding the optimal membrane may require experimenting with your specific protein on different membranes. Knowing the properties and the advantages and disadvantages to each membrane will help determine the best format for your application.
|Reprobe characteristics||Binding interactions||Binding capacity||Advantages||Disadvantages|
|Nitrocellulose||Can be stripped and reprobed||Hydrophobic and electrostatic||80–100 µg/cm2||Tendency to exhibit lower background||Can be brittle and fragile, which limits use in stripping and reprobing|
|PVDF||Can be stripped and reprobed||Hydrophobic||170–200 µg/cm2||Tendency to be more durable than nitrocellulose||Must be pre-wetted with methanol or ethanol prior to use|
|Nylon||Can be stripped and reprobed||Ionic, hydrophobic, and electrostatic||480 µg/cm2||High durability||Higher non-specific binding to strong anions|
Several different transfer buffers are used for wet transfer methods. The type of buffer used is dependent on the protein of interest, the gel buffering system and transfer method.
In most experiments, SDS is omitted from the western transfer buffer because the negative charge imparted to proteins can cause them to pass through the membrane. Typically, there is enough SDS associated with the proteins from SDS-PAGE separation to effectively carry them out of the gel and onto the membrane support. For proteins that tend to precipitate, the addition of low concentrations of SDS (<0.01%) may be necessary. It should be noted that adding SDS to the transfer buffer may require optimization of other transfer parameters (e.g., time, current) to prevent over-transfer of the proteins through the membrane (also known as "blow through").
Methanol is included in most transfer buffer formulations because methanol aids in stripping the SDS from proteins from separation by SDS-PAGE, increasing their ability to bind to support membranes. However, methanol can inactivate enzymes required for downstream analyses, and it can shrink the gel and membrane, which may increase the transfer time of large molecular weight proteins (150 kDa) with poor solubility in methanol. In the absence of methanol, though, protein gels may swell in low ionic strength buffers, and therefore it is recommended to pre-swell gels for 30 minutes to 1 hour to prevent band distortion.
Common transfer buffers for wet transfer
|Transfer Buffer||Formulation||Gel system||When to use|
|Towbin Transfer Buffer||25 mM Tris-HCl, 192 mM glycine, 20% (v:v) methanol, pH 8.3||Tris-glycine gels, Tricine gels|
|CAPS Transfer Buffer||10 mM CAPS, 10% (v:v) methanol, pH 10.5||Tris-glycine gels, Tricine gels||Target protein has pI >8.5; performing Edman protein sequencing|
|Bis-Tris Transfer Buffer||25 mM Bicine, 25 mM Bis-Tris (free base), 1 mM EDTA, 20% (v:v) methanol, pH 7.2||Bis-Tris gels, Tris-acetate gels, Tris-glycine gels||Need to limit protein modifications during transfer, performing Edman protein sequencing|
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