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Application-Specific PVDF Membranes: Immun-Blot PVDF Membrane for Western Blotting and Sequi-Blot PVDF Membrane for Protein Sequencing, Rev C

Eric Larson, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA


Introduction
PVDF membrane was originally introduced to protein use as an ideal medium for the harsh chemical environment of N-terminal, or Edman degradation, sequencing. Even though PVDF is very hydrophobic and requires a prewetting step in alcohol, its high protein binding capacity, target retention, and resistance to cracking have made it an appealing membrane for general laboratory techniques.

The two main applications for PVDF are N-terminal sequencing and immunoblotting, both of which benefit from the qualities of PVDF but rely on different features of the membrane. While sequencing work is concerned with retaining as much protein as possible, a western blot requires good signal retention with very low background. To provide the best membrane for each technique, Bio-Rad offers two grades of PVDF: Immun-Blot PVDF membrane for western blotting and Sequi-Blot PVDF membrane for protein sequencing.


Immun-Blot PVDF Membrane
The focus with a blotting membrane is on how well it delivers signal while resisting background and nonspecific binding. Immun-Blot PVDF membrane is ideal for chemiluminescent (Figure 1) and colorimetric (Figure 2) western blots because it very strongly retains target protein (140150 g protein/cm2 membrane) but resists background that can obscure highsensitivity detection. Immun-Blot PVDF membrane retains proteins in any transfer format: tank blotting, semi-dry blotting, and dot blotting all deliver excellent results. For proteins that are difficult to transfer, up to 0.1% SDS can be added to the transfer buffer without affecting the binding of the proteins to PVDF. The results are consistently clean, easy-to-read blots. The physical strength of Immun-Blot PVDF membrane means that it will not crack or tear during common handling, and will hold up under repeated stripping and reprobing applications.


Sequi-Blot PVDF Membrane
For protein sequencing applications, Sequi-Blot PVDF membrane is the best choice due to its extremely high protein binding capacity of 170200 g/cm2. This is the original Bio-Rad PVDF membrane, designed to withstand the conditions of N-terminal sequencing while providing the binding capacity to sequence even low-abundance samples. The table shows the increased protein recovery possible with Sequi-Blot PVDF membrane. Recovery of blotted proteins is typically in the range of 80100% of the initial sample load, resulting in higher initial coupling yields.

The binding efficiency of Sequi-Blot PVDF membrane is illustrated in Figure 3. This experiment showed that Sequi-Blot PVDF membrane is able to retain proteins that transfer through a competitors product.


Bibliography
Hulmes JD et al., Strategies for microcharacterization of proteins using direct chemistry on sequencer supports, in Techniques in Protein Chemistry (T Hugli, ed) Academic Press, San Diego, 716 (1989)

Matsudaira P, Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes, J Biol Chem 262, 10,035 10,038 (1987)

Speicher DW, Microsequencing with PVDF membranes: efficient electroblotting, direct protein adsorption and sequencer program modifications, in Techniques in Protein Chemistry (T Hugli, ed) Academic Press, San Diego, 2435 (1989)

Speicher DW et al., Electrotransfer and sequence enhancements improve low picomole sequence analysis using PVDF membranes without glass fiber filters, J Prot Chem 9, 254255 (1990)

Towbin H et al., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Natl Acad Sci USA 76, 43504354 (1979)

Yuen SW et al., Microanalysis of SDS-PAGE electroblotted proteins, Biotechniques 7, 7483 (1989)


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