Membrane Selection
Membrane Selection for Western Blotting
Polyvinylidene fluoride (PVDF) and nitrocellulose are the two membrane types most commonly used in Western blotting applications.
PVDF was first introduced as a substrate by Millipore Corporation in 1985. There are many advantages to electroblotting onto PVDF membranes rather than onto nitrocellulose membranes. PVDF membranes offer better protein retention, physical strength and broad chemical compatibility (Pluskal, et al., 1986). The higher mechanical strength and superior chemical resistance of PVDF membranes make them ideal for a variety of staining applications and reprobing in immunodetection. Another advantage of using PVDF membranes is that replicate lanes from a single gel can be used for various applications, such as staining with Coomassie™ Blue dye followed by band excision and N-terminal sequencing, proteolysis/peptide separation/internal sequencing, and immunodetection (Kurien, et al., 2003). Typical binding capacity of commercially available nitrocellulose membranes is 80–100 µg/cm2 while PVDF membranes offer a binding capacity of 100–200 µg/cm2.
In direct comparison of PVDF and nitrocellulose membranes in a serological assay for human immunodeficiency virus (HIV), PVDF membrane was shown to have better retention of total HIV antigens and improved detection of antibodies to glycosylated envelope antigens (Lauritzen and Pluskal, 1988).
For a comparision, see the table “Comparison of PVDF and nitrocellulose membrane attributes and applications” below.
Immobilon™PVDF Transfer Membranes
Millipore offers three PVDF membranes
- Immobilon-P membrane (0.45 µm) is a versatile substrate that is well suited for common immunoblotting applications</li>
- Immobilon-PSQ 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 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 transfer membranes are available in rolls and cut sheets. Millipore’s cuts are compatible with all the pre-cast gels and most commercially available gel running systems. See the table “Properties and applications of Immobilon PVDF transfer membranes” below. See the two subsequent tables to match Immobilon membrane cuts with the most commonly used electrophoresis systems and to match Immobilon membrane cut sizes with available pre-cast gels.
Comparison of PVDF and Nitrocellulose Membrane Attributes and Applications
| Attributes/Applications | Nitrocellulose | PVDF |
| Physical strength | Poor | Good |
| Protein binding capacity | 80- 100 µg/cm2 | 100- 300 µg/cm2 |
| Solvent resistance | No | Yes |
| Western transfer | Yes | Yes |
| Total protein stain | Colloidal gold Ponceau-S red Amido black India ink Syproâ blot stains | Colloidal gold Ponceau-S red Amido black India ink Coomassieä Blue dye |
| Detection | Chromogenic Chemiluminescent Fluorescent Radioactive | Chromogenic Chemiluminescent Fluorescent Chemifluorescent Radioactive |
| Double-blotting method | No | No |
| Rapid Immunodetection | No | Yes |
| Western reprobing | Yes | Yes |
| Edman sequencing | No | Yes |
| Amino acid analysis | Yes | Yes |
| Binding in the presence of SDS | Poor | Good |
| On-membrane digestion for mass spectrometry | No | Yes |
| Direct MALDI-TOF MS analysis | No | Yes |
| Data can be archived | No | Yes |
Properties and Applications of Immobilon PVDF Transfer Membranes
 | Immobilon-P transfer membrane | Immobilon-PSQ transfer membrane | Immobilon-FL transfer membrane |
| Description | Optimized to bind proteins transferred from a variety of gel matrices | Uniform pore structure results in superior binding of proteins with MW <20 kDa | Optimized for fluorescence immunodetection applications |
| Composition | PVDF | PVDF | PVDF |
| Pore size | 0.45µm | 0.2µm | 0.45 µm |
| Phobicity | Hydrophobic | Hydrophobic | Hydrophobic |
| Applications | Western blotting Binding assays Amino acid analysis N-terminal protein sequencing Dot/ slot blotting Glycoprotein visualization Lipopolysaccharide analysis Mass spectrometry | Low molecular weight western blotting Amino acid analysis Mass spectrometry N-terminal protein sequencing | Western blotting Dot/ slot blotting Fluorescence immunodetection |
| Detection methods | Chromogenic Chemifluorescent Chemiluminescent Radioactive | Chromogenic Fluorescent Chemifluorescent Chemiluminescent Radioactive | Fluorescent Chromogenic Chemifluorescent Chemiluminescent |
| Protein binding capacity | Insulin:160 µg/cm2 BSA: 215 µg/cm2 Goat IgG: 294 µg/cm2 | Insulin:262 µg/cm2 BSA: 340 µg/cm2 Goat IgG: 448 µg/cm2 | Insulin:155 µg/cm2 BSA: 205 µg/cm2 Goat IgG: 300 µg/cm2 |
| Compatible stains Reversible | Transillumination Ponceau-S CPTS Toluidine Blue Syproâ blot stains | Transillumination Ponceau-S CPTS Toluidine Blue Syproâ blot stains | Transillumination Ponceau-S CPTS Syproâ blot stains |
| Irreversible | Coomassieä Brilliant Blue dye Amido black India Ink Colloidal Gold | Coomassieä Brilliant Blue dye Amido black India Ink Colloidal Gold | Coomassieä Brilliant Blue dye Amido black |
Immobilon PVDF Transfer Membrane Cuts and Matching Electrophoresis Systems
| Manufacturer | Vertical Gel Box | Gel Size (cm) | Immobilon Size (cm) | Immbilon-P 0.45 µm | Immbilon-PSQ 0.2 µm | Immbilon-FL 0.45 µm* |
| GE Healthcare | SE 260 Mighty Small | 8 x 7 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| GE Healthcare | SE 260 Mighty Small | 8 x 9.5 | 8 x 10 | IPVH08100 | ISEQ08100 | |
| GE Healthcare | miniVE | 8 x 9.5 | 8 x 10 | IPVH08100 | ISEQ08100 | |
| GE Healthcare | miniVE | 10 x 10 | 10 x 10 | IPVH10100 | ISEQ10100 | IPFL10100 |
| GE Healthcare | SE 400 | 14 x 16 | 15 x 15 | IPVH15150 | ISEQ15150 | |
| GE Healthcare | SE 600 | 14 x 16 | 15 x 15 | IPVH15150 | ISEQ15150 | |
| GE Healthcare | SE 600 | 14 x 8 | 13.5 x 8.5 | IPVH08130 | ISEQ08130 | |
| Bio-Rad | Mini-PROTEAN 3 | 8.3 x 7.3 | 8.4 x7 | IPVH07850 | ISEQ07850 | IPFL00010 |
| Bio-Rad | Mini-PROTEAN 3 Dodeca | 13.3 x 8.7 | 13.5 x 8.5 | IPVH08130 | ISEQ08130 | IPFL20200 |
| Bio-Rad | Criterion, Criterion Dodeca | 13.3 x 8.7 | 13.5 x 8.5 | IPVH08130 | ISEQ08130 | |
| Bio-Rad | PROTEAN II xi | 16 x 16 | 15 x 15 | IPVH15150 | ISEQ15150 | |
| Bio-Rad | PROTEAN II xi | 16 x 20 | 26.5 x 375 | IPVH00010 | ISEQ00010 | |
| Bio-Rad | PROTEAN II XL | 19.3 x 18.3 | 20 x 20 | IPVH20200 | ISEQ20200 | |
| Bio-Rad | PROTEAN Plus Dodeca | 20 x 20.5 | 26 x 26 | IPVH304F0 | ISEQ304F0 | |
| Bio-Rad | Mini-PROTEAN II | 8.3 x 7.3 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Invitrogen | XCell Surelock Mini-Cell XCell6 MultiGel | 8 x 8 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Owl | P81 Puffin, P82 Wolverine | 10 x 10 | 10 x 10 | IPVH10100 | ISEQ10100 | IPFL10100 |
| Owl | P8DS Emperor Penguin | 10 x 10 | 10 x 10 | IPVH10100 | ISEQ10100 | IPFL10100 |
| Owl | P8DS Emperor Penguin | 10 x 10 | 10 x 10 | IPVH10100 | ISEQ10100 | IPFL10100 |
| Owl | P9DS Emperor Penguin | 8 x 10 | 8 x 10 | IPVH08100 | ISEQ08100 | IPFL10100 |
| Owl | P10DS Emperor Penguin | 16 x 16 | 15 x 15 | IPVH15150 | ISEQ15150 | IPFL10100 |
| Owl | P10DS Emperor Penguin | 20 x 20 | 20 x 20 | IPVH20200 | ISEQ20200 | IPFL20200 |
| Thermo Electron | EC120 | 8 x 7 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
*All sizes can be cut from rolls of Immobilon-FL membrane (IPFL00010).
Immobilon PVDF Transfer Membrane Cuts and Matching Pre-cast Gels
| Manufacturer | Precastl Gel Box | Gel Size (cm) | Immobilon Size (cm) | Immbilon-P 0.45 µm | Immbilon-PSQ 0.2 µm | Immbilon-FL 0.45 µm* |
| Bio-Rad | Ready Gel | 8.3 x 7.3 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Bio-Rad | Criterion | 13.3 x 8.7 | 13.5 x 8.5 | IPVH08130 | ISEQ08130 | |
| Bio-Rad | PROTEAN Ready Gel | 16 x 16 | 15 x 15 | IPVH15150 | ISEQ15150 | |
| Bio-Rad | PROTEAN Ready Gel | 19.3 x 18.3 | 20 x 20 | IPVH20200 | ISEQ20200 | IPFL20200 |
| Bio-Rad | PROTEAN Ready Gel | 20 x 20.5 | 26 x 26 | IPVH304F0 | ISEQ304F0 | |
| Cambrex | PAGEr | 9 x 10 | 8 x 10 | IPVH08100 | ISEQ08100 | |
| Cambrex | PAGEr | 10 x 10 | 10 x 10 | IPVH10100 | ISEQ10100 | IPFL10100 |
| Gradipore | MicroGel | 8 x 2.5 | 8 x 10 (cut in °) | IPVH08100 | ISEQ08100 | |
| Gradipore | igels | 8 x 5.8 | 8 x 10 (cut in ¥) | IPVH08100 | ISEQ08100 | |
| Gradipore | LongLife Gels | 8 x 5.8 | 8 x 10 (cut in ¥) | IPVH08100 | ISEQ08100 | |
| Invitrogen | NuPAGE | 8 x 8 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Invitrogen | Novex | 8 x 8 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Invitrogen | Zoom | 8 x 8 | 8.4 x 7 | IPVH07850 | ISEQ07850 | |
| Invitrogen | E-Gel | 13.5 x 10.8 | 13.5 x 8.5 | IPVH08130 | ISEQ08130 | |
| Pierce | Precise Protein Gels | 8 x 5.8 | 8 x 10 (cut in ¥) | IPVH08100 | ISEQ08100 | |
* All sizes can be cut from rolls of Immobilon-FL membrane (IPFL00010).
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