PVDF membrane

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). 

Transfer Thin-Sliced Tissue Section onto the PVDF Membrane... 

The last sample preparation method for IMS is the transfer of a tissue section onto the PVDF membrane. Proteins in the section can be transferred onto the PVDF membrane and then analyzed on the membrane. The advantage of this method is that the enzyme can be digested for MS" measurement, because the information on protein localization in the organization is fixed on the membrane.5,20 This technique can denature, reduce, and digest the proteins in the tissue section efficiently and remove the salt from the tissue. This increases the efficiency with which biological molecules are ionized, making it possible to obtain sensitive mass imaging spectra. 

Fig. 1.2 Protein blot analysis of human therapeutic protease inhibitor (HTPI) produced in alfalfa cell cultures using different promoters and subcellular targeting peptides as shown. Equal amounts of total soluble proteins from cell cultures were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinyldifluoride (PVDF) membrane. Monoclonal anti-HTPI IgGs were used for detection.

Fig. 1.3 Prediction of the most appropriate subcellular targeting strategies by agroinfiltration. The levels of an industrial enzyme (IE) are shown in agroinfiltrated and transgenic alfalfa leaves using different subcellular targeting peptides. Equal amounts of total soluble leaf proteins were separated by SDS-PAGE and blotted onto a PVDF membrane. Polyclonal anti-IE IgGs were used for detection.

Fig. 1.4 Protein blot analysis of C5-1 assembly in agroinfiltrated alfalfa leaves. Total leaf soluble proteins, extracted 4 days after infiltration were separated by SDS-PAGE under non-reducing conditions and blotted onto a PVDF membrane. Polyclonal antimouse IgGs were used for detection. Purified C5-1 was mixed with total soluble proteins from control infiltrated alfalfa leaves and loaded as a standard.

Proteins blotted on PVDF membranes are stainable with Coomassie Brilliant Blue R250, but a relative intense background remains, which does not influence, for example, amino acid sequence analysis. 

CPTS is also suitable for staining of proteins on PVDF membranes (see Protocol 2.4.5.1). 

In follow-up work, Kimura et al. (2005) classihed the removal of various pharmaceuticals, by a 0.4-pm PVDF membrane versus conventional activated sludge into three distinct categories (Table 5.5). A few other physical characteristics of those compounds are also included in Table 5.5 to give some additional information that may influence membrane performance. Note that all of the compounds in category 2 in Table 5.5 have Cl ion in their structure. The chloride ion is fairly common in PPCPs (Jjemba, 2006), and chloride-based compounds are usually quite recalcitrant (Eker and Kargi, 2006). 

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

HaloTag-frision proteins separated by SDS-PAGE were elec-trophoretically transferred onto a PVDF membrane using the transfer buffer with the semidry transfer device (2 mA/cm, 60 min). 

The resultant PVDF membrane was washed with TBS including 0.05% Tween-20 (TBST) for 10 min with gentle agitation (incubation was done with gentle agitation thereafter). 

On-blot digestion is another method used for proteins blotted onto nitrocellulose or PVDF membranes. Procedure is similar to in-gel digestion with the exception of elution from the membrane surface. 

PVDF film PVDF membranes PVDF monofilament... 

Follow the instructions of the suppliers of the blotting equipment for the electrophoretic transfer of proteins from SDS-containmg polyacrylamide gels to either nitrocellulose or PVDF membranes (see ref 4 and Chapter 20 for detailed instructions)... 

PVDF membrane (e.g, Immobilon PTM from Millipore Corp, Bedford, MA)... 

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