Types of Western Blot Transfer Buffers (2024)

To maintain conductivity and pH transfer buffers contain a conductive, strong buffering agent (for example, Tris, CAPS, or carbonate). In addition, alcohol (for example, methanol or ethanol) may be included in the transfer buffer to promote binding of proteins to membranes, and SDS may be added to promote elution of proteins from gels.

Regardless of the transfer buffer selected, when preparing and using transfer buffers:

• Do not use the same batch of transfer buffer more than once, as the buffer will likely lose its capacity to maintain a stable pH during transfer
• Do not dilute the transfer buffer; this will also decrease buffering capacity
• Do not adjust the pH of transfer buffers when not indicated, as this increases buffer conductivity, which is manifested by higher initial current output and decreased resistance

The most common transfers are from SDS-PAGE gels using the buffer systems originally described by Towbin (1979). Standard Towbin buffer contains 25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol and, occasionally, 0.025–0.1% SDS.

A buffer similar in composition to the standard Towbin buffer is the Bjerrum Schafer-Nielsen buffer (48 mM Tris, 39 mM glycine, pH 9.2, 20% methanol), which was developed for use in semi-dry applications.

CAPS-based transfer buffer (10 mM CAPS, pH 11, 10% methanol) may be preferable for transfers of high molecular weight proteins (for example, >150 kD) and in cases where the glycine component of Towbin buffer may interfere with downstream protein sequencing applications.

A unique feature of semi-dry blotting is the ability to use two different buffers during transfer, known as a discontinuous buffer system. In a semi-dry transfer, the buffer reservoirs are the filter paper on either side of the gel, which are independent (discontinuous). In a discontinuous system, methanol is included in the buffer on the membrane (anode) side of the blot assembly and SDS is used on the gel (cathode) side, taking advantage of the positive effects of each buffer component. A discontinuous buffer system using a Tris-CAPS buffer can greatly increase the efficiency of protein transfer by semi-dry blotting. This system uses 60 mM Tris, 40 mM CAPS, pH 9.6, plus 15% methanol in the filter paper on the anode side and 0.1% SDS on the cathode side. Concentrated, premixed anode and cathode buffers are available for purchase. For more information about the use of a discontinuous buffer system in semi-dry transfer, see Bio-Rad bulletin 2134.

In some cases, using a carbonate buffer (10 mM NaHCO₃, 3mM Na₂CO₃, pH 9.9, 20% methanol) may produce higher efficiency transfers and improve the ability of antibodies to recognize and bind to proteins. Carbonate buffer has also been recommended for the transfer of basic proteins (Garfin and Bers 1989).

The mobility of proteins during electrophoretic transfer from native gels will depend on the size and pI of the protein of interest relative to the pH of the buffer used.

• If the pI of the protein is greater than the pH of the transfer buffer, the protein carries a positive charge and will migrate toward the negative electrode
• If the pI of the protein is close to the pH of the transfer buffer, the migration of the protein out of the gel is decreased. Use a more basic or acidic buffer to increase protein mobility

Proteins in native gels, as well as acidic and neutral proteins, require buffers that do not contain methanol. Gels for isoelectric focusing, native PAGE, and those containing basic proteins or acid-urea may be transferred in 0.7% acetic acid. When using acetic acid for transfer, the proteins will be positively charged, so the membrane should be placed on the cathode side of the gel.

General guidelines for transfer buffer and membrane selection by gel type.

General guidelines for transfer buffer and membrane selection by application.

A Note Regarding SDS and Alcohol
SDS and alcohol play opposing roles in a transfer. SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gel but inhibits binding of the protein to membranes. In cases where certain proteins are difficult to elute from the gel, SDS may be added to the transfer buffer to improve transfer. SDS in the transfer buffer decreases the binding efficiency of protein to nitrocellulose membrane; PVDF membrane can be substituted for nitrocellulose when SDS is used in the transfer buffer. Addition of SDS increases the relative current, power, and heating during transfer and may affect the antigenicity of some proteins.

Alcohol (methanol or ethanol), on the other hand, removes the SDS from SDS-protein complexes and improves the binding of protein to nitrocellulose membrane but has some negative effects on the gel itself. Alcohol may cause a reduction in pore size, precipitation of some protein, and some basic proteins to become positively charged or neutral. All of these factors will affect blotting efficiency.

Note: Only high-quality, analytical grade methanol should be used in transfer buffer; impure methanol can increase transfer buffer conductivity and result in poor transfer.

Garfin DE and Bers G (1989). Basic aspects of protein blotting. In Protein Blotting: Methodology, Research and Diagnostic Applications, B.A. Baldo et al., eds. (Basel, Switzerland: Karger), pp. 5–41.

Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.