Immobilon^® PVDF membranes for Western blotting

Western blotting requires extensive, multi-parameter optimization and antibody-based detection protocols that consume valuable sample and reagents. Immobilon^® blotting membranes exhibit high protein binding capacity with superior retention across a broad range of protein sizes, including low-abundant proteins - minimizing time and conserving costly reagents while maximizing sensitivity and signal. We offer a wide selection of membrane chemistries and formats for your convenience.

Section overview

• Membrane selection for Western blotting
• Immobilon^® transfer membranes for Western blotting
• Immobilon^® NOW membrane format
• Factors affecting protein binding in Western blotting
• Binding differences between Immobilon^®-P and Immobilon^®-P^SQ transfer membranes
• Related products

Membrane selection for Western blotting

Membrane selection can influence the following factors in immunoblotting:

• Protein binding capacity
• Requirement for prewetting with alcohol
• Ability to perform multiple stripping and reprobing experiments
• Protein visualization
• Long-term blot storage
• Signal-to-noise ratio

Nitrocellulose and polyvinylidene fluoride (PVDF) are the most commonly used membranes for protein blotting.

1. Nitrocellulose is a versatile and reliable membrane well known for its performance in traditional Western blotting. It has a high protein binding capacity, low background noise, and is compatible with multiple detection methods and sample types.
2. PVDF membranes have a wider selection for different needs, such as low background for fluorescence and smaller pore size to capture smaller proteins. The higher mechanical strength of PVDF membranes also makes them ideal for reprobing or long-term membrane storage.

Both types of membranes offer high protein retention and broad chemical compatibility (Table 1). The typical binding capacity of nitrocellulose membranes is 200-250 μg/cm², while the binding capacity of PVDF membranes can range from 150-400 μg/cm² depending on the membrane type.

Immobilon^® transfer membranes for Western blotting

We offer five membranes:

• Immobilon^®-NC Nitrocellulose membrane (0.45 μm) is an excellent substrate for immunodetection and is compatible with standard blocking agents and detection protocols.
• Immobilon^®-P PVDF membrane (0.45 μm) is a versatile substrate that is well suited for common immunoblotting applications.
• Immobilon^®-E PVDF membrane (0.45 μm) wets out in water or buffer, eliminating the alcohol pre-wet step while retaining similar performance to Immobilon^®-P membranes.
• Immobilon^®-P^SQ PVDF membrane (0.2 μm) is ideal for protein sequencing and immunoblotting of low molecular weight proteins. It has a higher protein binding capacity and a higher retention than 0.45 μm membranes.
• Immobilon^®-FL PVDF membrane (0.45 μm) was developed for fluorescence-based immunodetection. It has very low background fluorescence across a wide range of excitation and emission wavelengths. 

Immobilon^® NOW membrane format

Providing the ultimate convenience in Western blotting, our Immobilon^® NOW Dispenser facilitates rapid, precise measurement and cutting of transfer membranes to standard mini and midi sizes. All Immobilon^® membrane types NC, P, E, P^SQ, FL, and E are available in the Immobilon^® NOW format. The Immobilon^® NOW format can be used in place of cut sheets when one dimension is 8.5 cm.

Factors affecting protein binding in Western blotting

At the molecular level, protein adsorption results, at least in part, from the interaction of hydrophobic amino acid side chains and hydrophobic domains with the polymer surface. Matsudaira observed an 80% decline in the sequencing efficiency of small peptides after hydrophobic residues were cleaved, presumably due to the washout of the peptide remnants.¹ Also, in peptide digestions, it has been observed that peptides characterized as hydrophobic often do not elute from the membrane as efficiently as more hydrophilic peptides.^{2, 3} McKeon and Lyman demonstrated that addition of Ca2+ ions to the transfer buffer enhanced the binding of calmodulin to the Immobilon^®-P transfer membrane.⁴ Binding of the calcium to the protein causes formation of a hydrophobic pocket in the molecule’s structure, leading to enhanced binding to the hydrophobic PVDF surface.

Different membranes require different treatments for wetting out depending on whether the membrane is hydrophobic or hydrophilic. Traditional PVDF membranes will not wet out in aqueous solutions and must first be wet in ≥50%(v/v) solution of alcohol such as methanol, ethanol, or isopropanol. Immobilon^®-E and Immobilon^®-NC are hydrophilic and can be wet out in an aqueous solution such as transfer buffer or water. For all membranes, complete wetting is evident by a change in the membrane’s appearance from opaque to semi-transparent.

Binding differences between Immobilon^®-P and Immobilon^®-P^SQ transfer membranes

After wetting, protein binding can be achieved by simply bringing the protein into contact with the membrane. Because binding occurs throughout the depth of the membrane, the binding capacity is determined by the internal surface area of the pores.⁵ Immobilon^®-P^SQ transfer membrane has approximately three times the internal surface area of Immobilon^®-P transfer membrane, resulting in higher adsorptive capacity (Table 2). The values listed in Table 2 represent upper limits for protein binding after saturation of the membrane surface in a nondenaturing buffer. However, the maximum binding that can be achieved will depend on the specific protocols employed, variations in the structural conformation of the proteins, the chemical nature of the buffers used, and the limitations of the methods used to apply the sample. An example of the binding difference between Immobilon^®-P and Immobilon^®-P^SQ transfer membranes is shown in Figure 2, where protein samples were electrotransferred from a polyacrylamide gel. A fraction of the proteins passed through the Immobilon^®-P transfer membrane and were captured on a second membrane placed behind the first one. In contrast, all of the proteins were bound to the Immobilon^®-P^SQ membrane without passing through it. In this case, the tighter pore structure and higher internal membrane surface area facilitated complete adsorption of all of the transferred protein. However, immunodetection on Immobilon^®-P^SQ transfer membrane can result in a higher background and can require more stringent washing conditions.

Thus, membrane choice is dictated by the goal of the experiment; use Immobilon^®-P transfer membrane for high-sensitivity detection of > 20 kDa proteins, but switch to Immobilon^®-P^SQ transfer membrane if smaller proteins are being analyzed or 100% protein capture is necessary for peptide sequencing.

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