Western blotting of proteins was introduced by Towbin et al. in 1979 and is now a routine 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. Western blotting can produce qualitative and semi-quantitative data on a protein of interest.
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Workflow diagram. Workflow of the tank electrotransfer of proteins for western blotting.
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). Alternatively, proteins can be separated by their isoelectric point (pI) using isoelectric focusing (IEF). After electrophoresis, the separated proteins are transferred, or "blotted", onto a solid support matrix, which is generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. In procedures where protein separation is not required, the proteins may be directly applied to the solid support by spotting the sample on the membrane using an approach called dot blotting.
In most cases, the membrane must be blocked to prevent nonspecific binding of the antibody probes to the membrane surface. The transferred protein is then complexed with an antibody and detection probe (e.g., enzyme, fluorophore, isotope). An appropriate method is then used to detect the localized probe to document the position 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 best results depend on the nature of the gel, the molecular weight of the proteins being transferred, the type of membrane and transfer buffers used, and the transfer method.
Watch this video on moving proteins from gel to membrane
Protein Transfer Technical Handbook
This 23-page handbook provides an in depth description of protein transfer, which is a vital step in western blotting. The transfer of proteins involves separation in a gel by electrophoresis to a solid support matrix so that specific proteins can be detected using immunodetection techniques. Thermo Fisher Scientific offers a complete array of products to support rapid and efficient protein transfer for western blotting. Our portfolio of high-quality protein transfer products unites membranes, buffers, stains, and molecular weight markers alongside a comprehensive choice of transfer devices designed to suit your needs and enable better quality western blot results.
Thermo Scientific blotting membranes may be available 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).
Following gel electrophoresis, there are a number of supports for protein transfer (including glass, plastic, latex and cellulose). The most common immobilization membranes are nitrocellulose, polyvinylidene difluoride (PVDF) and nylon. These membranes offer:
- A large surface area-to-volume area ratio
- A high binding capacity
- Extended storage of immobilized macromolecules
- Are easy to use
- Can be optimized for low background signal and reproducibility
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 to 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. However, traditional nitrocellulose membranes may be brittle and fragile, which limits their use for western blot experiments requiring stripping and reprobing procedures.
Use of nitrocellulose and PVDF membranes for western blotting. HeLa cell lysate was serially diluted and separated by SDS-PAGE electrophoresis. Proteins were then transferred from gel to nitrocellulose or PVDF membrane. Finally, blots were probed with respective specific antibodies, and target proteins were detected using a Thermo Scientific Pierce Fast Western Blot Kit and imaged using the Thermo Scientific myECL Imager. The data show a highly efficient transfer and detection of low, medium and high molecular weight proteins.
PVDF membranes are highly hydrophobic and must be pre-wetted with methanol or ethanol prior to submersion in transfer buffer. 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. In these applications, binding likely occurs via dipole and hydrophobic interactions. PVDF membranes have a protein binding capacity of 170-200 480 µg/cm2 and offer better retention of adsorbed proteins than other supports because of the greater hydrophobicity. PVDF is also less brittle than nitrocellulose and may be used for western blot experiments requiring stripping and reprobing procedures.
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 offer advantages in western blot experiments requiring stripping and reprobing procedures. A significant drawback to using nylon membranes for blotting applications is the possibility for nonspecific binding and strong binding to anions like SDS and Ponceau S.
Watch this video on preparing blots to reprobe
Common transfer buffers used for western blotting are:
- Towbin system buffer (25 mM Tris-HCl pH 8.3, 192 mM glycine, 20% (v:v) methanol)
- CAPS buffer system (10 mM CAPS pH 10.5, 10% (v:v) methanol)
In most experiments, SDS should be 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 in SDS-PAGE gels 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 "blowout").
Methanol in the transfer buffer aids in stripping the SDS from proteins in SDS-PAGE gels, 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 (150,000 Da) 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.
Watch this video on how to perform a western transfer
There are four major ways to transfer macromolecules from SDS-PAGE or native gels to nitrocellulose, PVDF or nylon membranes:
- Diffusion blotting
- Vacuum blotting
- Tank (wet) electrotransfer
- Semi-dry electrotransfer
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 the 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.
Wet electroblotting (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 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. Transfers are typically performed with an ice pack and at 4°C to mitigate the heat produced. A high field option exists for 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, blowout) 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).
Tank transfer apparatus for western blotting. Schematic showing the assembly of a typical 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)
For semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes in a semi-dry transfer apparatus. For this semi-dry transfer, it is very important that the gel is pre-equilibrated in transfer buffer. To maximize the current passing through the gel instead of around the gel, the amount of buffer available during transfer is limited to that contained in the sandwich, so it is helpful if the extra-thick filter paper (~3 mm thickness) and membrane are also sufficiently soaked in buffer. Likewise, it is key that the filter paper sheets and membrane are cut to the size of the gel.
One to four gels may be rapidly electroblotted to membranes. Methanol may be included in the transfer buffer, but other organic solvents, including aromatic hydrocarbons, chlorinated hydrocarbons and acetone, should not be used to avoid damage to the semi-dry blotter. 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. Methanol-free transfer buffers are recommended to reduce transfer time to 7 to 10 minutes. Transfer efficiencies of 60 to 80% may be achievable for proteins between 14 and 116 kDa.
Semi-dry electroblotting transfer. The Thermo Scientific Pierce 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 Pierce 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.
Comparison of 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 effort. For convenience and time savings, some researchers have switched to semi-dry blotting, but often at a loss of transfer quality. However, dry electroblotting offers both high quality transfer combined with speed as well as convenience since added buffers are not required for dry electroblotting.
Comparison of dry and semi-dry electroblotting. SW480 human colon cancer cell lysate was serially diluted (Lanes 1–6; 0.0625 µg, 0.215 µg, 0.25 µg, 0.5 µg, 1.0 µg, and 2.0 µg) and separated by SDS-PAGE alongside 5 µl Invitrogen SeeBlue Plus2 Pre-stained Protein Standard (Lanes 7 and 12) and 0.5 µl, 1.0 µl, 2.0 µl, and 4.0 µl Invitrogen MagicMark XP Western Protein Standard. The (A) iBlot dry transfer method produced a higher transfer efficiency compared with (B) the semi-dry transfer method.
Comparison of dry and wet electroblotting. SW480 human colon cancer cell lysate was serially diluted (Lanes 2–7; 0.0625 µg, 0.215 µg, 0.25 µg, 0.5 µg, 1.0 µg, and 2.0 µg) and separated by SDS-PAGE alongside 5 µl SeeBlue Plus2 Pre-stained Protein Standard (Lanes 1 and 8) and 0.5 µl, 1.0 µl, 2.0 µl, and 4.0 µl MagicMark XP Western Protein Standard (Lanes 9–12). Compared with the wet transfer method, the iBlot dry transfer method produced a higher transfer efficiency.
- Towbin, et al. (1979) Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. PNAS 76:4350–4354.
- Kurien, B.T. and Scofield, R.H. (2009) Introduction to Protein Blotting. In: Protein blotting and detection: methods and protocols. New York: Humana Press. pp 9–22.
- Kurien, B.T. and Scofield, R.H. (2009) Non-electrophoretic Bi-directional Transfer of a Single SDS-PAGE Gel with Multiple Antigens to Obtain 12 Immunoblots. In: Protein blotting and detection: methods and protocols. New York: Humana Press. pp 55–65.
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- Karey KP, Sirbasku DA. (1989) Glutaraldehyde Fixation Increases Retention of Low Molecular Weight Proteins (Growth Factors) Transferred to Nylon Membranes for Western Blot Analysis. Anal. Biochem. 178:255–259.
- Peferoen, M. (1988) Vacuum Blotting: An Inexpensive, Flexible, Qualitative Blotting Technique. In: Walker, J.M., Ed. Methods in Molecular Biology-New Protein Techniques. New York: Humana Press. Vol. 3, pp 383–393.
- Gooderham, K. (1984) Transfer Techniques in Protein Blotting. In: Walker, J.M., Ed. Methods in Molecular Biology-Proteins. New York: Humana Press. Vol. 1, pp 165–177.
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- Tovey, E.R. and Baldo, B.A. (1987) Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis 8:384–387.
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