Membranes and Blotting Papers

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Overview

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.

Related Topics: Protein Blotting Methods, Protein Blotting Equipment, and Transfer Buffers.

Nitrocellulose and Supported Nitrocellulose

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.

Polyvinylidene Difluoride (PVDF) Membranes

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 and Immun-Blot LF PVDF for Western Blotting

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 for Protein Sequencing

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 Papers

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

Membrane/Filter Paper Sandwiches

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.

Related Content

Literature
Number Description Download
1529 Western Blotting Troubleshooting, Rev C Click to download
2032 Western Blotting Detection Reagents Brochure, Rev F Click to download
2895 Protein Blotting Guide, Interactive PDF, Rev B Click to download
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