Membranes and Filter Papers for Western Blotting

PVDF (polyvinylidene difluoride) and nitrocellulose membranes are used for transferring proteins following gel electrophoresis for subsequent immunodetection. Compare the various membranes available for western blotting and read about various considerations when choosing the right membrane for your experiment.

Protein transfer is easy and convenient with our pre-cut, pre-assembled western blot membrane/filter paper sandwiches designed to fit your gels. Alternatively, membrane rolls, sheets, and filters papers are available to enable individual sizing and assembly.

Wet tank transfer options

For rapid dry and semi-dry transfer systems, like the iBlot 3 Western Blot Transfer System and Power Blotter Semi-Dry Transfer System, specialized transfer stacks are available.

Rapid transfer system options

High-quality western blotting results can often be achieved using either nitrocellulose or PVDF membranes. Both types of membranes offer efficient protein transfer and strong protein binding, enabling consistent results and sensitive detection for chemiluminescent western blotting. However, this can be highly protein dependent. Certain proteins bind more tightly to one type of membrane over the other. Broad assertions, such as better performance based on high or low molecular weight proteins, can be misleading or inaccurate. A protein’s properties (i.e., charge, hydrophobicity, etc.) affect its ability to bind to membrane surfaces; so, finding the optimal membrane may require experimenting with your specific protein on various membranes. 

The detection method used can also impact the target signal and membrane background. In fluorescent western blotting, or when using fluorescent total protein normalization methods such as Invitrogen No-Stain Protein Labeling Reagent, exposure times are often limited due to membrane auto-fluorescence and background noise, which negatively impact the achievable limit of detection. Optimized low fluorescence PVDF membranes exhibit low auto-fluorescence across fluorescent channels enabling low background and high signal to noise, making them excellent for fluorescent-based western blotting applications.

Lastly, there are handling considerations that may influence the decision to use nitrocellulose or PVDF. PVDF membranes are generally considered more durable than nitrocellulose. However, PVDF requires an activation step to enable good protein binding. This step is usually performed through a 3–5-minute incubation in 100% methanol or ethanol. In contrast, nitrocellulose membranes do not require this pre-activation step but can be more brittle than PVDF and require more careful handing to avoid tearing. 

(TOP) 

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, and nucleic acids. These membranes are composed of nitrocellulose, a cellulose derivative that helps provide a robust and reliable medium for protein immobilization. Nitrocellulose membranes are compatible with colorimetric, chemiluminescent, and most fluorescent detection methods. Their ease of use and compatibility with various staining and detection protocols make these membranes a great choice for western blotting.

(TOP) 

PVDF (polyvinylidene difluoride) membranes are hydrophobic, microporous membranes that are an ideal choice for western blotting applications, as well as for amino acid analysis and protein sequencing of small amounts of proteins (as little as 10 pmoles). In western blotting, PVDF membranes bind proteins through hydrophobic and dipole interactions. 

PVDF membranes require an activation step prior to submersion in transfer buffer. This activation step is necessary due to the highly hydrophobic characteristics of PVDF. During the activation step, methanol or ethanol serves as a liaison between the non-polar membrane and the polar buffer solution, allowing the transfer buffer to interact with the membrane. 

(TOP) 

When performing fluorescent western blotting, standard PVDF membranes commonly exhibit high levels of autofluorescence, as can nitrocellulose membranes, particularly in lower-wavelength channels (i.e., UV, 488 and 550 nm). This is a significant disadvantage because the high level of fluorescent background can interfere with accurate detection and analysis. Therefore, the proper choice of membrane for fluorescent western blotting is crucial as it affects the usable fluorescent channels, detection limits, and overall accuracy.

Invitrogen low fluorescence PVDF membranes exhibit minimal auto-fluorescence across all fluorescent channels and result in reduced background compared to both nitrocellulose and standard PVDF membranes (Figure 1). Additionally, Invitrogen low fluorescence PVDF membranes feature a unique 0.3 µm pore size with high consistency and uniformity across the membrane surface, making them an excellent choice for high-quality fluorescent western blotting applications. 

Compared to other commercially available low fluorescence PVDF membranes, Invitrogen low fluorescence PVDF membranes offer exceptional performance when imaging in lower wavelength channels (Figure 2). Lower background fluorescence allows for more protein targets per blot, higher-quality data publication, and several additional benefits including:

• Higher signal-to-noise-ratio—decreasing background noise enhances signal clarity (Figure 3)
• More sensitive detection—reducing autofluorescence improves limit of detection
• Enhanced multiplexing—improves results across multiplexing experiments, particularly when leveraging low-wavelength channels

Western blotting filter papers are pre-cut cotton sheets for wet or semi-dry, passive or electrophoretic transfer of proteins from polyacrylamide gels (SDS-PAGE) to PVDF, nitrocellulose, or other membranes. Sheets of filter paper are necessary components of transfer sandwiches and cassettes that typically must be assembled for various kinds of protein or nucleic acid, gel-to-membrane, transfer protocols. High-quality western blotting filter papers are available as standard-thickness and extra-thick sheets that are pre-cut for convenience with mini gels and midi gels. Choose a paper with the thickness and dimensions appropriate for specific gel sizes and device platforms or cassettes.

(TOP)