Nylon membrane

The amplification products were electrophoresed on agarose gels. Blots were performed according to protocols supplied with Hybond - N-nylon membrane (Amersham). 

Because membrane filtration is the only currently acceptable method of sterilizing protein pharmaceuticals, the adsorption and inactivation of proteins on membranes is of particular concern during formulation development. Pitt [56] examined nonspecific protein binding of polymeric microporous membranes typically used in sterilization by membrane filtration. Nitrocellulose and nylon membranes had extremely high protein adsorption, followed by polysulfone, cellulose diacetate, and hydrophilic polyvinylidene fluoride membranes. In a subsequent study by Truskey et al. [46], protein conformational changes after filtration were observed by CD spectroscopy, particularly with nylon and polysulfone membrane filters. The conformational changes were related to the tendency of the membrane to adsorb the protein, although the precise mechanism was unclear. 

This protocol permits detection of 10 pmol of target DNA dotted on a nylon membrane after hybridization with the d(G)30 probe [15]. The background chemiluminescence caused by nonspecific binding of the probe in the hybridization buffer to the membrane is negligible in this assay system. However, both the target DNA and the cDNA probe bound to the membrane are detected solely... 

Figure 11 Chemiluminescent detection for membrane hybridization of unmodified DNA target by derivatization reaction with TMPG. Procedure [15] A portion of the DNA solution is spotted on a nylon membrane. The target DNA is hybridized to its cDNA probe having a -(G)15TT(G)15TT at its 3 terminus in a hybridization buffer (pH 7.0) at 42°C for 2 h. After washing, the membrane is moistened with sodium phosphate solution (pH 10) for a few seconds, and then immersed in 0.2 M TMPG dissolved with dimethyl sulfoxide for 0.5 min at ambient temperature. The moist membrane is then dipped in dimethyl-formamide for a few seconds, and the luminescence is detected for 0.5 min.

All water samples were collected in amber-polyethylene terephtalate (PET) bottles and were kept at 4°C during shipment. Upon reception in the laboratory, samples were vacuum filtered through 1 pm glass fiber filters, followed by 0.45 pm nylon membrane filters, and were stored in the dark at —20°C until analysis. 

Transfer by blotting to a nylon membrane allows the heat treatment involved in hybridisation to take place. Southern blotting technique. 

Eiltration with nylon membrane filters sonication of the filters with three volumes of toluene rotavaporation and reconstitution in a 1.5 mL of toluene-methanol (2 1, v/v) mixture... 

Moreover, the unique adsorption properties of GEC allowed the very sensitive electrochemical detection of DNA based on its intrinsic oxidation signal that was shown to be strongly dependent of the multi-site attachment of DNA and the proximity of G residues to GEC [100]. The thick layer of DNA adsorbed on GEC was more accessible for hybridization than those in nylon membranes obtained with genosensors based on nylon/GEC with a changeable membrane [99,101,102]. Allhough GEC has a rough surface, it is impermeable, while nylon is more porous and permeable. DNA assays made on an impermeable support are less complex from a theoretical standpoint [7] the kinetics of the interactions are not compUcated by the diffusion of solvent and solutes into and out of pores or by multiple interactions that can occur once the DNA has entered a pore. This explained the lower hybridization time, the low nonspecific adsorplion and the low quantity of DNA adsorbed onto GEC compared to nylon membranes. 

Nitrocellulose and nylon membranes have been widely used in the production of macroarrays (arrays with probe sites of diameter 0.5 to 1 mm), but not so much in the production of microarrays (feature size of 25 to 200 xm) because of a lack of spot resolution (see Sect. 5.2, Spotting of Capture Probes). These membranes exhibit lateral wicking characteristics and the probe therefore tends to spread out from the point of apphcation. Casting of these membranes onto the surface of glass slides is a solution to this problem [28]. 

For longer double-stranded nucleic acids such as cDNA [ 200 bp (base pairs) to 1500 bp], the positively charged nylon membrane easily sequestered... 

Membranes such as NC supported on glass may be more applicable for protein microarrays than glass substrates. Supported charged nylon membranes for microarrays are currently entering the marketplace as well. The essential ingredient for protein is water. Protein hydration reduces the likelihood for surface denaturation. Hydrophilic membranes allow proteins to... 

Hornberg, J.J., de Hass, R.R., Dekker, H., and Lankelma, J., Analysis of multiple gene expression array experiments after repetitive hybridizations on nylon membranes, Biotechniques, 33, 108-117, 2002. 

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