PVDF filters

Spot proteins at high density on PVDF filters ... 

Filter aqueous polyphenol ic extract (sample) through a 0.45- im PVDF filter. Load a known volume of filtered extract onto cartridges. 

Filter the aqueous polyphenolic extract with a 0.45-jam PVDF filter and adjust the pH to 7.0 with 5 N NaOH. 

In contrast, the filtration method is more expensive (filters cannot be reused) but works with smaller net weights (i.e. sample volumes) and is usually more rapid, provided the cells are not slimy and do not clog the filter. Nylon or PVDF filters withstand the pre-drying procedure excellently whereas modified cellulose filters lose their mechanical robustness. 

On the other hand, PAMPA is a purely artificial method and PAMPA membranes do not reassemble real lipid bilayer structures as barriers for permeation but much thicker barriers. The thickness and material of the supporting PVDF filters also influences artificially the permeation of compounds depending on the lipophilicity of the compounds more than the thin polycarbonate filter does in CACo2 experiments. Also the best choice of membrane constituents for PAMPA experiments is still under investigation and it seems that it will depend a lot on the goal of the PAMPA experiment which membrane is used (e.g. blood brain barrier permeation or intestinal absorption). One has to take into account that PAMPA today is a summary term on a lot of different methods applied in different laboratories using different membrane constituents, sink conditions, permeation times etc., which makes inter laboratory comparison difficult. 

Another assay that has gained popularity and acceptance for the evaluation of permeability is the parallel artificial membrane permeability assay (PAMPA). Earlier versions of this system coated polyvinylidene fluoride (PVDF) filter plates with artificial membrane using dioleoyl-sn-glycerol-3-phosphocholine (Chen et al., 2008). Several companies attempted to develop this technology in the late 1990s with limited success (Kansy et al., 1998). These earlier versions suffered poor correlation to cell models, poor correlation to human absorption, and poor reproducibility. More modem systems have been developed with different lipid formulations and solvation techniques that seem to correlate better with Caco-2 and human data and are more reproducible (Chan et al., 2005). Some companies use the PAMPA as a tier 1 prescreen discovery... 

A PVDF membrane filter has been shown to remove >10 particles of vims for vimses >50 nm independent of fluid type (8). Vimses smaller than 50 nm are not removed as efficientiy but are removed in a predictable manner which correlates to the vims particle size. The chemistry of the suspending fluid affects titer reduction for vimses <50 nm owing to other removal mechanisms, such as adsorption, coming into play. The effects of these other mechanisms can be minimized by using filtration conditions that minimize adsorption. 

PVDF-based microporous filters are in use at wineries, dairies, and electrocoating plants, as well as in water purification, biochemistry, and medical devices. Recently developed nanoselective filtration using PVDF membranes is 10 times more effective than conventional ultrafiltration (UF) for removing vimses from protein products of human or animal cell fermentations (218). PVDF protein-sequencing membranes are suitable for electroblotting procedures in protein research, or for analyzing the phosphoamino content in proteins under acidic and basic conditions or in solvents (219). 

Figure 7.7. Protein microarray on polyvinylidene difluoride filters (PVDF). Proteins are gridded on filter and probed with an antibody to a gene of interest. Protein-ligand interactions could be detected using a labeled ligand as a probe.

Zhu and coworkers used hydrophilic filters (low protein binding PVDF) in their model [179]. This change in the setup resulted in a significant transport time reduction to 2 h (compared with more than 10 h for a hydrophobic filter) without loss of information for low permeability compounds. 

Filter paper (six sheets) and PVDF membrane are soaked in transfer buffer for 30 min. 

Amido black dye Two years later, Grassman and Hanning developed another organic dye to be used on filter paper after electrophoresis amido black stain. It has moderate sensitivity. Today, amido black dye is used for colorimetric determination of electroblotted proteins on PVDF (poly-vinylidene difluoride) and nitrocellulose membranes. 

Cut the PVDF transfer membrane about 0.5 to 1 cm larger than the gel. Cut a piece of Whatman no. 1 filter paper -0.5 cm larger than the PVDF membrane. 

Figure B3.2.2 Electroblotting with a tank transfer unit. The polyacrylamide gel containing the protein(s) to be transferred is placed on the smooth side of the polyethylene sheet (or filter paper sheets) and covered with the PVDF membrane and then a single sheet of filter paper. This stack is sandwiched between two fiber pads and secured in the plastic gel holder cassette. The assembled cassette is then placed in a tank containing transfer buffer. For transfer of negatively charged protein, the membrane is positioned on the anode side of the gel. Charged proteins are transferred electrophoretically from the gel onto the membrane.

Briefly wet the filter paper with transfer buffer and cover the PVDF membrane. Smooth gently to remove any air bubbles. Place the other fiber pad on top of the membrane and close the holder. 

A fully automated system uses either cDNA fragments of known genes or libraries of oligonucleotides, which are then spotted in duplicate onto hybridization filters (nylon membranes or PVDF) with a grid for guidance. When nylon membranes are used, these filters in microarrays can be used several times. There are two possible ways of applying the DNA to the filter (Cahill, 2001) ... 

All the filters used must comply with the demands made by regulatory authorities. In sterilizable systems, hydrophobic diaphragm filters made of either polyvinylidene fluoride (PVDF) or polytetrafluoroethylene (PTFE) with a diaphragm pore width of... 

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