Charged nylon

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... 

TTBS (nitrocellulose or PVDF) or TBS (neutral or positively charged nylon see recipes for both solutions)... 

For nitrocellulose and PVDF 0.1% (v/v) Tween 20 in TBS (TTBS see recipe). For neutral and positively charged nylon Tris-buffered saline (TBS see recipe) containing 10% (w/v) nonfat dry milk. 

Set up a capillary transfer pyramid using negatively charged nylon membrane and 10X SSC as the transfer buffer, Allow to transfer overnight. Fix the DNA to the membrane by baking or ultraviolet crosslinking... 

Store substrate at 4°C in the dark (stock solution 10 mg/ml = 23.5 mM), Do not use nitrocellulose membranes but Biodyne, Hybond N or Magnagraph nylon membranes use unpowdered gloves. Use probes at less than 20 ng/ml (Chapter 8). Signals on nitrocellulose are much weaker and require special blocking and enhancer substances (Nitro-Block Sapphire or Emerald from Tropix). Best results are on positively charged nylon also PVDF with Nitro-Block. 

Charged nylon membranes (Nytran, Zeta-Probe, Magnagraph ... 

PVDF membranes have been used occasionally (Hicks and Vecoli, 1987 Williams, 1990). They have binding characteristics similar to charged nylon although the binding is hydrophobic, with possible electrostatic interactions since PVDF polymers have a strong dipole character. This may be the reason why PVDF functions very well in alkaline conditions. 

Heparin (50 mg in 4 X SSPE or 4 X SSC) is also used as blocking agent at 50 [xg/ml or at 500 xg/ml if dextran sulfate is present (Singh and Jones, 1984). Lebacq et al. (1988) observed that heparin (a polyanionic mucopolysaccharide) lowered the general background significantly on positively charged nylon when used at 3.5 mg/ml. 

Disassemble the apparatus when wells are empty and UV irradiate the samples (better and more convenient than baking). Baking or UV irradiation of alkaline-immobilized DNA on charged nylon membranes may be counterproductive. When using UV irradiation for the first time, it is recommended to optimize the time required (usually 1-5 min) for best binding to the damp membranes (e.g., uncovering a successive dot every 30 s) always use the same distance from the source. Store as in A7. 

As a general rule, prehybridization conditions (buffer and temperature) are, except for ohgonucleotide probes and charged nylon, identical to those of hybridization although the time is shorter (usually fast but prolonged prehybridization does not harm). Most (pre)hybridization buffers are variants of two basic buffers, one with formamide and one without. Formamide allows the use of lower incubation temperatures and an easy adjustment of the stringency and is generally used, or even essential, for RNA hybridization or for nonradioactive probes. It is recommended, particularly when formamide is used, to include a buffer, such as sodium/potassium phosphate, Tris-HCl, or PIPES-NaOH, or to use SSPE instead of... 

This method is suited for nitrocellulose, nylon and Immobilon (= PVDF) however, positively charged nylon permits direct transfer of DNA in strong base (shorten denaturation step 1 to 30 min and omit step 2). 

Cut positively charged nylon membrane to the size of the gel and float on water until completely wetted from underneath and immerse in 10 X SSC. 

Remove excess liquid and place wetted charged nylon membrane (as in D3) on gel followed by 3MM paper and a blotting stack. As in bidirectional transfer, the solution in the gel is the only transfer medium. 

Northern analysis (Alwine et al., 1977, 1979) of RNA after electrophoretic fractionation was initially carried out on diazotized (DBM) paper, but its detection limit was only about 500 pg RNA (using a probe of 10 cpm/ xg). Subsequently, Thomas (1980, 1983) succeeded in detecting less than 1 pg on nitrocellulose. Diazotized (APT) paper was also found to be superior to DBM paper (Seed, 1982), however nitrocellulose and charged nylon (Reed and Mann, 1985) have become the preferred membranes. RNA can be detected at less than 0.01% (10 xg loaded) on nitrocellulose or, particularly on nylon which usually has an increased background, however. Nylon membranes are required if reprobing is desired. 

Buluwela et al. (1989) established a rapid procedure in which lysis, DNA denaturation and fixation is achieved in a single step using positively charged nylon membranes for lifts and a microwave oven... 

After gel electrophoresis, the DNA fragments are transferred to a charged nylon membrane. 

This has a higher binding capacity than nitrocellulose and is more rugged. It is quite resistant to chemical attack. Positively charged nylon membranes (PCM) have a high protein binding capacity > 500 pg/cml... 

The most significant claim is for the removal of "pyrogens" (endotoxins). Table 2.6 17 compares the pyrogen removal efficiency for a positively charged nylon 0.2 ju pore size membrane, Zetapor , and a conventional cellulosic 0.22 ju membrane. Normally, a 10,000 molecular weight cut-off UF membrane is required to remove pyrogens. (These membranes have an equivalent pore-size of 30 A or 0.003 ju). As expected, the conventional MF membrane shows no retention whatsoever, but the positively charged membrane shows better than 97% retention. 

Blot membranes consist of nitrocellulose (BA 85 from Schleicher and Schiill), polyvinyliden difluoride (PVDF) (Immobilon from Millipore), positively charged nylon ( nylon) (Zetaprobe from Bio-Rad), or glass fiber coated with polybrene (GF/C from Whatman). The membranes bind the proteins through hydrophobic (nitrocellulose) or hydrophobic and ionic interaction ( nylon, polybrene-coated glass fiber). Even peptides with only 20 amino acids stiU stick to nitrocellulose. 

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