Mansfield M A
Millipore Corporation, Bedford, Massachusetts 01730, USA.
Anal Biochem. 1995 Jul 20;229(1):140-3. doi: 10.1006/abio.1995.1391.
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.
此处呈现的数据（图2，表2）清楚地表明，转铁蛋白的免疫检测并不需要对Immobilon-P进行润湿处理。可以推断，即使聚偏二氟乙烯（PVDF）的周边区域保持疏水状态，固定化的转铁蛋白在分子水平上也能经历足够的再水化过程，从而使抗体能够识别表位。疏水印迹法与采用槽式转移和半干式转移制备的印迹以及底物5-溴-4-氯-3-吲哚磷酸/硝基蓝四唑（BCIP/NBT）和4-氯-1-萘酚（4CN）均兼容。此外，在疏水印迹方案中，抗转铁蛋白抗血清的结合特异性未发生改变。鉴于现有的封闭剂、抗体和可视化系统种类繁多，可能需要针对特定的试剂组合进行优化。最重要的参数将是所使用的封闭剂和去污剂的浓度，因为根据化学性质和浓度的不同，它们中的任何一种都可能导致膜在孵育过程中出现润湿现象。该方法不适用于硝酸纤维素膜，因为在其制造过程中专门使用了表面活性剂来使其在水性缓冲液中发生润湿。
