Table 1.
Properties of membranes used for Southern blotting and hybridization
| Type of membrane | |||
|---|---|---|---|
| Property | Nitrocellulose | Neutral nylon | Charged nylon |
| Capacity (µg nucleic acid/cm2) | 80–120 | ∼100 | 400–500 |
| Size of nucleic acid required for maximal binding | >400 bp | >50 bp | >50 bp |
| Transfer buffer | High ionic strength at neutral pH | Low ionic strength over a wide range of pH | |
| Immobilization | Baking at 80°C under vacuum for 2 h | Baking at 70°C for 1 h; no vacuum required | |
| or | |||
| mild alkali | |||
| or | |||
| UV irradiation at 254 nm; damp membranes are generally exposed to 1.6 kJ/m2; dried membranes require 160 kJ/m2 | |||
| Commercial products | Hybond-N | Hybond-N+ | |
| GeneScreen | Zeta-Probe | ||
| Nytran+ | |||
| GeneScreen Plus | |||
-
Polyvinylidene difluoride (PVDF) membranes are not routinely used for northern or Southern transfers. However, PVDF membranes, by virtue of their higher mechanical strength and greater capacity to bind proteins, are preferred to nitrocellulose for western blotting. Nylon membranes should not be used for western blotting because the level of nonspecific absorption of immunological probes is unacceptably high.
