Proteins are transferred from the gel to blotting membranes to make them amenable to immunodetection during western blotting. A variety of membrane types is available, each offering key attributes to suit particular experimental conditions. The physical properties and performance characteristics need to be evaluated when selecting a membrane for a specific application. This section provides an overview of blotting membranes offered by Bio-Rad and guidance on how to choose the best membrane for your applications.
Nitrocellulose was one of the first membranes used for western blotting and is still a popular membrane for this procedure. Protein binding to nitrocellulose is instantaneous, nearly irreversible, and quantitative to 80–100 µg/cm2. Nitrocellulose is easily wetted in water or transfer buffer and is compatible with a wide range of protein detection systems. Unsupported nitrocellulose is innately fragile and is not recommended for stripping and reprobing.
Supported nitrocellulose is an inert support structure with nitrocellulose applied to it. The support structure gives the membrane increased strength and resilience. Supported nitrocellulose can withstand reprobing and autoclaving (121°C) and retains the ease of wetting and protein binding features of nitrocellulose.
PVDF membranes are an ideal support for N-terminal sequencing, amino acid analysis, and immunoassay of blotted proteins (western blotting). PVDF retains proteins during exposure to acidic or basic conditions and in the presence of organic solvents. Greater protein retention during sequencing manipulations enhances the likelihood of obtaining information from rare, low-abundance proteins by increased initial coupling and higher repetitive yields. In addition, PVDF membranes exhibit better binding efficiency of electroblotted material in the presence of SDS in the transfer buffer.
PVDF membranes must be wetted in 100% methanol prior to use, but once wet may be used with a transfer buffer that contains no methanol. Bio-Rad offers PVDF membranes specifically designed for protein sequencing and for immunodetection. Both are available in precut sheets, rolls, and sandwich formats.
Immun-Blot PVDF membranes have a strong binding capacity of 150–160 µg/cm2 (roughly twice that of nitrocellulose), will not rack or tear in common handling, and can withstand repeated stripping and reprobing.
Immun-Blot LF PVDF membranes combine the advantages of Immun-Blot PVDF membranes with low autofluorescence properties across a wide range of excitation and emission wavelengths. This low autofluorescence allows longer exposure times without increasing background fluorescence levels, allowing fluorescent detection of faint signals and maximizing the detection capabilities of the imaging system.
Sequi-Blot PVDF membrane withstands the conditions of N-terminal sequencing while providing the binding capacity to sequence even low-abundance samples.
Guide to protein blotting membranes.
|Membrane||Pore Size||Binding Capacity (µg/cm2)||Compatible Detection Method||Notes|
|Nitrocellulose||0.45 µm 0.2 µm||80–100||Colorimetric Chemiluminescence Chemifluorescence Fluorescence Radioactive||General-purpose protein blotting membrane|
|Supported nitrocellulose||0.45 µm 0.2 µm||80–100||Colorimetric Chemiluminescence Chemifluorescence Fluorescence Radioactive||Pure nitrocellulose cast on an inert synthetic support; increased strength for easier handling and for reprobing|
|Immun-Blot PVDF||0.2 µm||150–160||Colorimetric Chemiluminescence Radioactive||High mechanical strength and chemical stability; recommended for western blotting|
|Immun-Blot LF PVDF||0.45 µm||155–300||Colorimetric Chemiluminescence Chemifluorescence Fluorescent||High mechanical strength and chemical stability; low autofluorescence; recommended for western blotting using fluorescent detection|
|Sequi-Blot™ PVDF||0.2 µm||170–200||Colorimetric Radioactive||High mechanical strength and chemical stability; recommended for protein sequencing|
Blotting filter paper, made of 100% cotton fiber, provides a uniform flow of buffer through the gel and contains no additives that might interfere with the transfer process. Precut filter paper is available in a wide range of convenient sizes to eliminate waste and save time. Extra thick absorbent filter paper is recommended for semi-dry transfers because of its additional fluid capacity.
Guide to precut membranes and filter paper.
|Blotting Cells||Precut Membranes||Precut Blot Filter Papers|
|Mini Trans-Blot® cell||7 x 8.5 cm||7.5 x 10 cm|
|Criterion™ blotter||8.5 x 13.5 cm||9.5 x 15.2 cm|
|Trans-Blot® cell||13.5 x 16.5 cm||15 x 20 cm|
|Trans-Blot® Plus cell||26.5 x 28 cm||26.5 x 28 cm|
|Trans-Blot® SD cell||7 x 8.5 cm
11.5 x 16 cm
15 x 15 cm
15 x 9.2 cm
20 x 20 cm
|15 x 15 cm (extra thick)|
|Trans-Blot® Turbo™||7 x 8.5 cm and 8.5 x 13.5 cm
Transfer packs include precut
membrane and filter paper
|Bio-Dot® apparatus||9 x 12 cm||N/A|
|Bio-Dot SF apparatus||9 x 12 cm||11.3 x 7.7 cm|
Precut and preassembled sandwiches save time and effort during western blot preparation. In Bio-Rad's membrane sandwiches, a precut membrane (nitrocellulose or PVDF) and two sheets of 100% cotton-fiber thick filter paper are preassembled into a blotting membrane/filter paper sandwich.