Western Blotting Membranes

Western blotting, (or immunoblotting) involves the separation of a protein mixture by gel electrophoresis followed by subsequent electrotransfer to a suitable membrane. Western blot membrane selection affects experimental workflow and results due to differences in these properties:

  • Protein binding capacity
  • Protein retention during incubation and wash steps
  • Requirement for prewetting with alcohol
  • Ability to perform multiple stripping and reprobing experiments
  • Protein visualization options
  • Long-term blot stability
  • Signal-to-noise ratio

PVDF vs Nitrocellulose Membranes

The first protein electroblotting experiments were performed using Millipore® nitrocellulose membranei, as the Western blotting method was derived from an earlier technique for identification of DNA sequences called Southern blotting. Immobilon® PVDF membranes were developed specifically for Western blotting a few years later. The higher mechanical strength, superior chemical resistance, and increased protein retention of PVDF membranes generally provide improved signal in immuoblotting applications and make them ideal for stripping and reprobing of a single blot with multiple antibodies (Table 1). In contrast to nitrocellulose, PVDF membranes are hydrophobic in nature and (except for Immobilon®-E) must be wetted with alcohol prior to use.

Table 1: Nitrocellulose vs PVDF membrane attributes

Attributes/Applications Nitrocellulose PVDF
Physical strength Poor Good
Protein binding capacity 100 – 200 µg/cm2 150 – 450 µg/cm2
Solvent resistance No Yes
Western transfer Yes Yes
Wets out with water and aqueous transfer buffers Yes No*
Total protein stain Colloidal gold Colloidal gold
  Ponceau-S red Ponceau-S red
  Amido black Amido black
  India ink India ink
  Sypro® blot stains Sypro® blot stains
    Coomassie® Blue dye
Detection Chromogenic Chromogenic
  Chemiluminescent Chemiluminescent
  Fluorescent Fluorescent
  Radioactive Radioactive
Western reprobing Yes Yes
Blot can be archived No Yes

*Except for Immobilon®-E

Immobilon® PVDF Transfer Membranes

We offer four PVDF membranes for protein blotting applications:

  • Immobilon®-P membrane is a versatile substrate, well-suited for common immunoblotting applications.
  • Immobilon®-E membrane offers similar performance to Immobilon®-P. Immobilon®-E features a transient, hydrophilic coating that allows it to be easily wetted with water and standard buffers, eliminating the need for prewetting with alcohol.
  • Immoblion®-PSQ membrane is ideal for protein sequencing and immunoblotting of low molecular weight proteins, due to its higher protein binding capacity and retention.
  • Immobilon®-FL membrane was developed for fluorescence-based immunodetection, with very low background fluorescence across a wide range of excitation and emission wavelengths.

Table 2: Properties and applications of Immobilon® PVDF transfer membranes

  Immobilon®-P Immobilon®-E Immobilon®-PSQ Immobilon®-FL

Description

Optimized to bind proteins transferred from a variety of gel matrices. First PVDF transfer membrane and the most cited product in its class. Good choice for standard Western blotting applications with proteins >20 kDA. Transient hydrophilic coating allows use without alcohol prewet. Uniform pore structure results in superior binding of proteins with MW <20 kDa. Optimized for fluorescence immunodetection applications. Low background fluorescence for visible and infrared probes.
Composition PVDF PVDF with hydrophilic coating PVDF PVDF
Pore size 0.45 µm 0.45 µm 0.2 µm 0.45 µm
Phobicity Hydrophobic Hydrophobic Hydrophobic Hydrophobic
Applications • Western blotting
• Binding assays
• Amino acid analysis
• N-terminal protein sequencing
• Dot/slot blotting
• Glycoprotein visualization
• Lipopolysaccharide analysis
• Mass spectrometry
• Western blotting • Low molecular weight Western blotting
• Amino acid analysis
• N-terminal protein sequencing
• Mass spectrometry
• Western blotting
• Dot/slot blotting
• Fluorescence immunodetection
Detection methods • Chemifluorescent
• Chemiluminescent
• Chromogenic
• Radioactive
• Chemifluorescent
• Chemiluminescent
• Chromogenic
• Radioactive
• Chemifluorescent
• Chemiluminescent
• Chromogenic
• Fluorescent
• Radioactive
• Chemifluorescent
• Chemiluminescent
• Chromogenic
• Fluorescent
• Radioactive
Protein binding capacity • Goat IgG: 294 µg/cm2
• BSA: 215 µg/cm2
• Insulin: 160 µg/cm2
• Goat IgG: 294 µg/cm2 • Goat IgG: 448 µg/cm2
• BSA: 340 µg/cm2
• Insulin: 448 µg/cm2
• Goat IgG: 300 µg/cm
• BSA: 205 µg/cm2
• Insulin: 300 µg/cm2
Compatible stains reversible • Transillumination
Ponceau-S
• Toluidine blue
• Sypro® blot stains
• Transillumination
• Ponceau-S
• Toluidine blue
• Sypro® blot stains
• Transillumination
• Ponceau-S
• Toluidine blue
• Sypro® blot stains
• Transillumination
• Ponceau-S
• Toluidine blue
• Sypro® blot stains
Compatible stains irreversible Amido black
• India ink
• Colloidal gold
• Coomassie™ Brilliant Blue dye
• Amido black
• India ink
• Colloidal gold
• Coomassie™ Brilliant Blue dye
• Amido black
• India ink
• Colloidal gold
• Coomassie™ Brilliant Blue dye
• Amido black
• India ink
• Colloidal gold

Protein Binding to PVDF Membranes

As a hydrophobic polymer, PVDF will not wet directly in aqueous solutions. To use PVDF-based membranes (other than Immobilon®-E ) in aqueous buffers, they must first be wet in a ≥ 50% (v/v) alcohol (i.e. methanol, ethanol, isopropanol) solution. Complete wetting is evident by a change in the membrane’s appearance from opaque to semi-transparent. After wetting, the alcohol must be removed by rinsing in water, and then the membrane can be directly equilibrated in transfer buffers.

Immobilon®-E wets out in water or standard transfer buffers, allowing users to skip the prewetting and subsequent water rinse steps. After that point, Immobilon®-E performs much like Immobilon®-P in terms of protein binding, staining and immunodetection. If the membrane dries out during later processing steps, Immobilon®-E must be wetted with alcohol prior to the addition of aqueous buffers.

Factors Affecting Protein Binding

At the molecular level, protein adsorption primarily stems from the interaction of hydrophobic amino acid side chains and hydrophobic domains with the polymer surface. Matsudairaii observed an 80% decline in sequencing efficiency of small peptides after cleaving hydrophobic residues, presumably due to washout of the peptide remnants. During peptide digestions, it has also been observed that peptides characterized as hydrophobic often do not elute from the membrane as efficiently as hydrophilic peptidesiiiiv. McKeon and Lymanv demonstrated that the binding of calmodulin to Immobilon®-P membranes could be enhanced by the addition of Ca2+ ions to the transfer buffer, due to the formation of a hydrophobic pocket in the protein molecular structure.

Protein Binding Differences between Immobilon®-P and Immobilon®-PSQ Transfer Membranes

Figure 1. Prolonged electrotransfer of proteins using Immobilon®-P and Immobilon®-PSQ transfer membranes. Molecular weight standards (lanes 1,3,5,7) and calf liver lysate (lanes 2,4,6,8) were transferred to Immobilon®-P or Immobilon®-PSQ membranes by the tank transfer method and stained with Coomassie® Blue. A sheet of Immobilon®-PSQ transfer membrane was placed behind the primary membranes to capture proteins that passed through them. (Lanes 5 and 6 behind Immobilon®-P; lanes 7 and 8 behind Immobilon®-PSQ.)

Protein binding can be achieved on a wet membrane by simply bringing the protein into contact with the membrane. Because binding occurs throughout the depth of the membrane, binding capacity is determined by the internal pore surface area (Mansfield, 1994). Pores in Immobilon®-PSQ transfer membrane have approximately three times more internal surface area than Immobilon®-P transfer membrane, resulting in higher adsorptive capacity (Table 2). The values listed in Table 2 represent upper limits for protein binding after membrane surface saturation in a non-denaturing buffer. In all applications, Immobilon®-PSQ transfer membrane can be expected to bind more protein than Immobilon®-P transfer membrane. However, maximum protein binding, for either membrane, depends on the specific protocols employed, due to 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®-PSQ transfer membranes is shown in Figure 1, 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 proteins bound to the Immobilon®- PSQ membrane, without passing through. In this case, the tighter pore structure and higher internal surface area of polymer facilitated complete adsorption of all transferred protein. However, immunodetection on Immobilon®-PSQ transfer membrane can result in higher background noise and can require more stringent washing conditions. Thus, membrane choice is dictated by your experimental goals. We recommend using Immobilon®-P transfer membrane for the high-sensitivity detection of >20 kDa proteins and switching to Immobilon®-PSQ transfer membrane if smaller proteins are being analyzed or 100% protein capture is necessary for peptide sequencing.

Immobilon® membranes for Western Blotting

Immobilon®-P PVDF Membrane, 0.45 µm pore size

Catalog Number Pore Size Filter Diameter (⌀) Filter Color Filter Surface Pack Size
IPVH00010 0.45 µm 26.5cm x 3.75m White Plain 1 roll
IPVH00005 0.45 µm 26.5cm x 1.875m White Plain 1 roll
IPVH07850 0.45 µm 7cm x 8.4cm White Plain 50 sheets
IPVH08100 0.45 µm 8cm x 10cm White Plain 10 sheets
IPVH08130 0.45 µm 8.5cm x 13.5cm White Plain 10 sheets
IPVH09120 0.45 µm 9cm x 12cm White Plain 10 sheets
IPVH10100 0.45 µm 10cm x 10cm White Plain 10 sheets
IPVH15150 0.45 µm 15cm x 15cm White Plain 10 sheets
IPVH20200 0.45 µm 20cm x 20cm White Plain 10 sheets
IPVH304F0 0.45 µm 26cm x 26cm White Plain 10 sheets
IPVH85R 0.45 µm 8.5 cm x 10 m White Plain 1 roll

Immobilon®-E PVDF Membrane, 0.45 µm pore size

Catalog Number Pore Size Filter Diameter (⌀) Filter Color Filter Surface Pack Size
IEVH85R 0.45 µm 8.5 cm x 10 m White Plain 1 roll
IEVH00005 0.45 µm 26.5cm x 1.875m White Plain 1 roll
IEVH07804 0.45 µm 7cm x 8.4cm White Plain 4 sheets
IEVH07850 0.45 µm 7cm x 8.4cm White Plain 50 sheets
IEVH08100 0.45 µm 8cm x 10cm White Plain 10 sheets
IEVH09120 0.45 µm 9cm x 12cm White Plain 10 sheets
IEVH10100 0.45 µm 10cm x 10cm White Plain 10 sheets

Immoblion®-PSQ PVDF Membrane, 0.2 µm pore size

Catalog Number Pore Size Filter Diameter (⌀) Filter Color Filter Surface Pack Size
ISEQ00010 0.2 µm 26.5cm x 3.75m White Plain 1 roll
ISEQ00005 0.2 µm 26.5cm x 1.875m White Plain 1 roll
ISEQ07850 0.2 µm 7cm x 8.4cm White Plain 50 sheets
ISEQ08100 0.2 µm 8cm x 10cm White Plain 10 sheets
ISEQ08130 0.2 µm 8.5cm x 13.5cm White Plain 10 sheets
ISEQ09120 0.2 µm 9cm x 12cm White Plain 10 sheets
ISEQ10100 0.2 µm 10cm x 10cm White Plain 10 sheets
ISEQ15150 0.2 µm 15cm x 15cm White Plain 10 sheets
ISEQ20200 0.2 µm 20cm x 20cm White Plain 10 sheets
ISEQ26260 0.2 µm 26cm x 26cm White Plain 10 sheets

Immoblion®-FL PVDF Membrane, 0.45 µm pore size

Catalog Number Pore Size Filter Diameter (⌀) Filter Color Filter Surface Pack Size
IPFL00010 0.45 µm 26.5cm x 3.75m White Plain 1 roll
IPFL00005 0.45 µm 26.5cm x 1.875m White Plain 1 roll
IPFL07810 0.45 µm 7cm x 8.4cm White Plain 10 sheets
IPFL10100 0.45 µm 10cm x 10cm White Plain 10 sheets
IPFL20200 0.45 µm 20cm x 20cm White Plain 10 sheets