Blotting includes various methods for transferring biological molecules (e.g., proteins, nucleic acid fragments) from a gel matrix to a membrane support for the subsequent detection of those molecules, and Western blotting is the method used for immunodetection of detect proteins.

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Western blotting was introduced by Towbin et al.1 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.

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, and 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.

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Western Blotting Handbook and Troubleshooting Guide

The "Thermo Scientific Pierce Western Blotting Handbook and Troubleshooting Guide" (2014) details each step of the Western blotting process with technical information and products for transfer, blocking, washing, antibodies, substrates, film and stripping buffer, including Pierce Fast Western products. You will want to keep this booklet close at hand because it also includes protocols, references and a troubleshooting guide.

Blotting Membranes

Blotting membranes may be available in either sheets or rolls and commonly have a thickness of 100µm with typical pore sizes of 0.1, 0.2 or 0.45µm. Most proteins can be successfully blotted using a 0.45µm pore size membrane, while a 0.1 or 0.2µm 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 have the following characteristics:

  • A large surface area-to-volume area ratio
  • A high binding capacity
  • Provide the extended storage of immobilized macromolecules
  • Are easy to use
  • Can be optimized for low background signal and reproducibility

Nitrocellulose Membranes

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, glycoproteins or nucleic acids. 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 proteins with higher molecular weights. Nitrocellulose membranes are not optimal for electrophoretic transfer of nucleic acids, as the high salt concentrations that are required for efficient binding will effectively elute some or all of the charged nucleic acid fragments.

PVDF Membranes

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; binding likely occurs via dipole and hydrophobic interactions. PVDF membranes offer a better retention of adsorbed proteins than other supports because of the greater hydrophobicity. PVDF is also less brittle than nitrocellulose.

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Nylon Membranes

Charged nylon (polyamide) membranes bind by ionic, electrostatic and hydrophobic interactions, and many of the charged nylons contain quaternary amines that form stable linkages with nucleic acids under alkaline conditions. While not always required, nucleic acids are typically fixed to the membrane by UV-crosslinking, while uncharged nylon membranes rely on hydrophobic interactions that are formed when nucleic acids are dried on to the membrane surface. 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. Membranes with a positive zeta charge are recommended for blotting applications with negatively-charged nucleic acids, such as electrophoretic mobility shift assays (EMSA).

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Transfer Buffers

Common 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 shrinks 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.

Transfer Methods

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

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.3 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.4Vacuum 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 30-65%, 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.5 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 gels 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.8 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 (0.45µm).

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.

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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 60-80% may be achievable for proteins between 14 and 116 kDa.

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Recommended References for Western blotting

  1. Towbin, et al. (1979). Electrophoretic Transfer of Proteins from Polyacrylamide Gels to Nitrocellulose Sheets: Procedure and Some Applications. PNAS. 76: 4350-4354.
  2. Kurien, B.T. and Scofield, R.H. Introduction to Protein Blotting. In Protein blotting and detection: methods and protocols. Humana Press: New York, NY, 2009; 9-22.
  3. Kurien, B.T. and Scofield, R.H. Non-electrophoretic Bi-directional Transfer of a Single SDS-PAGE Gel with Multiple Antigens to Obtain 12 Immunoblots. InProtein blotting and detection: methods and protocols. Humana Press: New York, NY, 2009; 55-65.
  4. Westermeier, R., et al. Blotting. In Eletrophoresis in Practice. A Guide to Methods and Applications of DNA and Protein Separations, 4th ed. Wiley-VCH: New York, NY, 2005; 67-80.
  5. Peferoen, M. Vacuum Blotting: An Inexpensive, Flexible, Qualitative Blotting Technique. In Methods in Molecular Biology-New Protein Techniques. Walker, J.M., Ed. Humana Press: New York, NY, 1988, Vol. 3, pp 383-393.
  6. Gooderham, K. Transfer Techniques in Protein Blotting. In Methods in Molecular Biology-Proteins. Walker, J.M., Ed. Humana Press: New York, NY, 1984, Vol. 1, pp 165-177.
  7. Khyse-Andersen, J. (1984). Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Biochem. Biophys. Meth. 10: 203.
  8. 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.