Multiple probing of an immunoblot membrane using a non-block technique: advantages in speed and sensitivity.
Multiple probing of an immunoblot membrane using a non-block technique: advantages in speed and sensitivity.
复制标题
使用非封闭技术对免疫印迹膜进行多次探测：速度和灵敏度方面的优势。
DOI：
10.1006/abio.1999.4453
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Imboden,JB
中科院分区：
文献类型：
--
作者：
Sadra,A;Cinek,T;Imboden,JB
Here we show a modification of an existing nonblock technique in immunoblotting (1). Our method incorporates three cycles of methanol–water hydration of the membrane allowing multiple erasure and probing of the same blot. This modification also improves the background obtained with each probing. In a side-byside comparison, we also demonstrate the relative superiority of the non-block technique over the conventional method in preserving the integrity of proteins on a blot after multiple stripping of the membrane. The non-block technique takes advantage of the inherent hydrophobicity of PVDF in bypassing the blocking steps required for the conventional Western blotting technique (1, 2). The wash steps are also shortened from multiple minutes to a few seconds (1). This technique does not work for nitrocellulose membranes because they do not maintain hydrophobicity during the incubation and wash steps (1). We performed a side-by-side comparison of both the non-block and conventional Western techniques in our protocol. Protein samples were solubilized by boiling in SDS sample buffer and were subjected to SDS–polyacrylamide gel electrophoresis according to Laemmli (3). The proteins were then transferred onto PVDF membranes (Immobilon-P)(Millipore, Bedford, MA) using a published protocol (4). The conventional and non-block Western techniques described here use identical incubation and blocking solutions. The incubation buffers were made in TBS-T2 (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.04% Tween 20). The blocking agents were either 2% BSA or 2% nonfat milk in TBS-T as specified. The sources for the antibodies used are published (5). Additional antibodies used were for human GRB2 (C-23) and human ZAP-70 (LR)(Santa Cruz, CA) along with human p38 MAPK (9212)(New England BioLabs, Beverly, MA). For the conventional technique, following protein transfer, the PVDF membrane was allowed to air-dry for 15 min. Afterwards, it went through a methanol–water hydration, followed by blocking of the membrane in 2% BSA–TBS-T (for antiphosphotyrosine probing) or 2% nonfat milk–TBS-T (for other probings) for 1 h at room temperature. The blocked membrane was then incubated for 1 h with the primary antibody solution at room temperature. The membrane was washed twice for 15 min each in TBS-T and then incubated with the secondary antibody solution for 30 min. The membrane was washed four times for 15 min with TBS-T prior to detection of the immunoblot signals with enhanced chemiluminescence (ECL) using Renaissance Western Plus reagent from NEN Life Sciences (Boston, MA). For the non-block technique, after the transfer of proteins onto the PVDF membrane, the membrane was air-dried for 15 min and went through three cycles of methanol–water hydration. This was done to drive out any residual SDS in the membrane. SDS can add unwanted background in the subsequent probing of the membrane. The blotted membrane was then left in methanol for 2 min and allowed to dry in an incubator for 10 min at 37 C. It was essential to completely dry the PVDF membrane because any loss of hydrophobicity of the membrane during the subsequent antibody incubations could cause unwanted background. There was no blocking step in this method. After the membrane was dried, it was incubated with the primary antibody solution (made in 2% nonfat milk–TBS-T) for 1 h at room temperature. Gentle shaking was used for the incubations, otherwise the membrane could become wet. The membrane was then washed twice with TBS-T for 20 s each, followed by incubation with the secondary antibody solution …
