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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-P^SQ 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.

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

	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

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

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