Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking.

Abstract

The data presented here (Fig. 2, Table 2) clearly demonstrate that wetting of Immobilon-P is not required for immunodetection of transferrin. It can be inferred that the immobilized transferrin undergoes sufficient rehydration at the molecular level to permit epitope recognition by the antibodies, even when the surrounding areas of PVDF remain hydrophobic. The hydrophobic blot procedure was compatible with blots prepared by both tank and semidry transfer and with the substrates BCIP/NBT and 4CN. Additionally, the binding specificity of antitransferrin antiserum was not altered in the hydrophobic blot protocol. Given the diversity of blocking agents, antibodies, and visualization systems available, optimization for specific reagent combinations may be necessary. The most important parameters will be the concentration of blocking agents and detergents used since either, depending on chemical properties and concentration, may cause the membrane to wet out during incubation. This procedure is not applicable to nitrocellulose membranes since surfactants are used in their manufacture specifically to cause wetting in aqueous buffers.