Western blot transfer from an SDS-PAGE gel is a method that can be used to detect individual proteins in a given complex sample. The denatured proteins are resolved by electrophoresis then transferred out of the PAGE gel and onto a microporous membrane, where they are located by stains or by “probing” with specific detection reagents, such as antibodies to the analyte of interest.
The two most common membrane types used for western blots are nitrocellulose and polyvinylidene fluoride (PVDF). Nitrocellulose was one of the first membrane types used for western blotting. Its advantages are that it is easily wetted and it produces good immunoblotting results. PVDF membranes were developed in 1985 and exhibit improved protein retention under harsh conditions (i.e., in the presence of organic solvents or under acidic or basic conditions). The greater mechanical strength of PVDF membranes is an asset when handling the membrane compared to nitrocellulose. In addition to its chemical stability, PVDF offers advantages when stripping and reprobing in immunodetection applications.
Three different PVDF membranes with characteristics tailored to specific uses are available from Pall Life Sciences:
FluoroTrans Membrane is a PVDF microporous membrane with a rated pore size of 0.2µm. It offers higher protein adsorption capacity and retention than any other commercially available membrane. Protein immobilized on FluoroTrans membrane is not easily removed, even with strong chaotropic agents. The high adsorption capacity, coupled with the high protein retention and resistance to chemical solvents, make this membrane the “gold standard” for use with N-terminal sequencing using the Edman chemistry.
Applications: N terminal protein sequencing, protein dot, or slot blotting
Advantages: High binding capacity, lowest burn-through, chemical resistance, high tensile strength, and low fluorescence back-ground
Tip: Some high background staining may be experienced when using total protein stains. This is presumably a consequence of the high internal surface area and adsorption potential afforded by such a material.
FluoroTrans W Membrane is a membrane that has been optimized for use in Western transfer applications. Sacrificing little in terms of binding capacity and retention, this membrane shows very low levels of protein “burn-through” during transfer. FluoroTrans W membrane yields very high sensitivity, excellent resolution, and very low background levels with all detection systems including total protein stains.
Applications: Protein transfers, protein dot or slot blots, and detection with immunostaining
Advantages: Highest sensitivity, low background, low burn-through, high binding capacity, chemical resistance, and high tensile strength
BioTrace PVDF Membrane with a rated pore size of 0.45 µm performs especially well with chemiluminescent and colorimetric detection systems. It is highly resistant to organic solvents and aggressive aqueous solutions. BioTrace PVDF membrane is recommended for all protein transfers.
Applications: Protein transfers, protein dot, or slot blots
Advantages: Highest sensitivity, low background, chemical resistance, and high tensile strength
Pall Life Sciences offers protocols for western blotting with FluoroTrans and BioTrace PVDF membranes.
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