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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
  • 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
Syproa blot stains
Colloidal gold
Ponceau-S red
Amido black
India ink
Coomassiea 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
Syproa blot stains
Transillumination
Ponceau-S
CPTS
Toluidine Blue
Syproa blot stains
Transillumination
Ponceau-S
CPTS
Syproa blot stains
Irreversible Coomassiea Brilliant Blue dye
Amido black
India Ink
Colloidal Gold
Coomassiea Brilliant Blue dye
Amido black
India Ink
Colloidal Gold
Coomassiea 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).

출처: http://www.millipore.com