Transfer Buffers

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Overview

Transfer buffers must enable both effective elution of proteins from the gel matrix and binding of the protein to the membrane. While different gel types and blotting applications call for different transfer buffers, the choice of buffer also depends on the membrane being used and the physical characteristics of the protein of interest. This section provides an overview of different transfer buffers, such as Tris/Glycine, CAPS, Dunn carbonate buffers and discontinuous buffer systems. It also provides general guidelines for selection of buffers and membranes according to gel types and applications.

Related Topics: Protein Blotting Methods, Protein Blotting Equipment, Membranes and Blotting Papers, and Transfer Conditions.

Considerations for Using Transfer Buffers

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

Towbin and Bjerrum Schafer-Nielsen Buffers

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 Buffer

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.

Discontinuous Tris-CAPS Buffer System (for Semi-Dry Transfer)

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.

Dunn Carbonate Buffer

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).

Other Buffers

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.

Gel Type Transfer Buffer Membrane Notes
SDS-PAGE Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen Nitrocellulose, supported nitrocellulose, or PVDF (0.45 or 0.2 µm) Tank blotting or semi-dry blotting
Tris-Tricine Towbin, CAP Nitrocellulose, supported nitrocellulose, or PVDF
(0.2 µm)
Tank blotting recommended; needs high-capacity, small pore-size membrane; pH of buffer may be critical
Two-dimensional Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen Nitrocellulose, supported nitrocellulose, or PVDF (0.45 or 0.2 µm) Tank blotting or semi-dry blotting
Native, nondenaturing Depends on pH of gel buffer and pI of protein of interest Nitrocellulose or PVDF (0.45 or 0.2 µm) Tank blotting recommended; temperature regulation may be needed to maintain activity
Acid urea 0.7% acetic acid Nitrocellulose
(0.45 or 0.2 µm)
Tank blotting or semi-dry blotting; use acid-gel transfer protocol (membrane toward cathode)
Isoelectric focusing 0.7% acetic acid Nitrocellulose, supported nitrocellulose, or PVDF (0.45 or 0.2 µm) Tank blotting or semi-dry blotting; use acid-gel transfer protocol (membrane toward cathode)

 

 

General guidelines for transfer buffer and membrane selection by application.

Application Transfer Buffer Membrane Notes
Protein sequencing Towbin*, CAPS Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank blotting recommended
High molecular weight proteins Towbin with SDS Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank or rapid semi-dry blotting recommended; needs high-capacity, small pore-size membrane; pH of buffer may be critical
Small proteins and peptides Towbin, CAPS Nitrocellulose,
0.2 µm, or PVDF
Tank or rapid semi-dry blotting recommended; pH of buffer may be critical
Basic proteins (pI >9) in denaturing gels CAPS, carbonate, Bjerrum Schafer-Nielsen Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank blotting, semi-dry blotting, or rapid semi-dry blotting
Basic proteins (pI >9) in native or nondenaturing gels 0.7% acetic acid Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank blotting recommended
Glycoproteins Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen nondenaturing gels Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank blotting or semi-dry blotting
Proteoglycans Towbin, Bjerrum Schafer-Nielsen Nitrocellulose, 0.45 or 0.2 µm, or PVDF Tank blotting or semi-dry blotting
*Towbin buffer may be used for protein sequencing but extra care must be exercised to rinse Tris and glycine from the membrane after transfer

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.

References

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.

Related Content

Literature
Number Description Download
2895 Protein Blotting Guide, Interactive PDF, Rev B Click to download
2032 Western Blotting Detection Reagents Brochure, Rev F Click to download
1529 Western Blotting Troubleshooting, Rev C Click to download
2134 Increased Transfer Efficiency Using a Discontinuous Buffer System With the Trans-Blot SD Cell, Rev B Click to download
Number Description Options
6211 Transfer Buffers Formulation Click to download
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