（五）免疫反应
（1）将膜用TBS从下向上浸湿后，移至含有封闭液的平皿中，室温下脱色摇床上摇动封闭1h。
（2）将一抗用TBST稀释至适当浓度（在1.5ml离心管中）；撕下适当大小的一块儿保鲜膜铺于实验台面上，四角用水浸湿以使保鲜膜保持平整；将抗体溶液加到保鲜膜上；从封闭液中取出膜，用滤纸吸去残留液后，将膜蛋白面朝下放于抗体液面上，掀动膜四角以赶出残留气泡；室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10min。
（3） 同上方法准备二抗稀释液并与膜接触，室温下孵育1～2h后，用TBST在室温下脱色摇床上洗两次，每次10min；再用TBS洗一次，10min，进行化学发光反应。
（六）化学发光，显影，定影
（1）将A和B两种试剂在保鲜膜上等体积混合；1min后，将膜蛋白面朝下与此混合液充分接触；1min后，将膜移至另一保鲜膜上，去尽残液，包好，放入X-光片夹中。
（2）在暗室中，将1×显影液和定影液分别到入塑料盘中；在红灯下取出X－光片，用切纸刀剪裁适当大小（比膜的长和宽均需大1cm）；打开X-光片夹，把X－光片放在膜上，一旦放上，便不能移动，关上X-光片夹，开始计时；根据信号的强弱适当调整曝光时间，一般为1min或5min，也可选择不同时间多次压片，以达最佳效果；曝光完成后，打开X-光片夹，取出X-光片，迅速浸入显影液中显影，待出现明显条带后，即刻终止显影。显影时间一般为1～2min（20～25℃），温度过低时（低于
16℃）需适当延长显影时间；显影结束后，马上把X-光片浸入定影液中，定影时间一般为5～10min，以胶片透明为止；用自来水冲去残留的定影液后，室温下晾干。
应注意的是：显影和定影需移动胶片时，尽量拿胶片一角，手指甲不要划伤胶片，否则会对结果产生影响。
（七） 凝胶图象分析
将胶片进行扫描或拍照，用凝胶图象处理系统分析目标带的分子量和净光密度值。
Western blot analysis-1 Cells were cultured in 199 medium with 10% fetal
bovine serum. After digestion with 0.25%trypsin and 0.2%EDTA, the cells
were collected and were washed three times with ice-cold PBS, then were
lysed in buffer(50mM Tris-HCl, pH8.0,150mM NaCl, 100μg/ml PMSF,
1%TritonX-100) for 30 min at ice. After removal of cell debris by
centrifugation(12,000g,5 min), the protein concentration of lysates was
meatured by Bradford method. 50μg proteins of different groups boiled
for 5 min in sample buffer and were separated in 10% SDS-PAGE and
transferred onto nitrocellulose membrane (Bio-Rad). Nonspecific
reactivity was blocked by incubation overnight at 4℃ in buffer(10mM
Tris-HCl, pH7.5,150mM NaCl, 2%Tween-20, 4% bovine sreum albumin).The
membrane was then incubated with primary antibody. The secondary
antibody was used to detected bound primary antibody. Reactive protein
was detected by ECL chemiluminescence system (Amersham pharmacia
biotech).
Western blot analysis-2 The protein expression ezrin was detected by
Western blot method. Confluent 150 cm2 flasks of cells were washed three
times with ice-cold PBS, then lysed in buffer(50mM Tris-HCl,
pH8.0,150mM NaCl, 100μg/ml PMSF, 1%TritonX-100) for 30 min at ice. After
removal of cell debris by centrifugation(12,000g,5 min), 50?g of each
lysate sample was boiled for 5 min in sample buffer and were separated
by 10% SDS-PAGE and transferred onto nitrocellulose membrane (Pall
Corporation). Nonspecific reactivity was blocked in 5% nonfat dry milk
in TBST(10mM Tris-HCl, pH7.5,150mM NaCl, 0.05%Tween-20) for 1h at room
temperature. The membrane was then incubated with monoclonal antibody of
mouse anti human ezrin p81(Maixin-Bio) followed by reaction with
anti-mouse IgG-HRP antibody. Reactive protein was detected by ECL
chemiluminescence system (Santa Cruz).
ezrin蛋白表达通过 Western blot进行检测。已达融合生长的单层细胞用冷PBS洗三次后，加入裂解液(50mM Tris-HCl,
pH8.0,150mM NaCl, 100μg/ml PMSF, 1%TritonX-100)冰上裂解30min，12,000g,5
min离心去除细胞碎片后，取50?g样品与上样缓冲液混合，煮沸5min，进行10% SDS-PAGE电泳，转硝酸纤维素膜(Pall
Corporation)。将膜在含5%脱脂奶粉的TBST(10mM Tris-HCl, pH7.5,150mM NaCl,
0.05%Tween-20)中室温下封闭1h，随后加入鼠抗人ezrin一抗(Maixin-Bio)，抗鼠IgG-HRP二抗，ECL化学发光试剂检测(Santa
Cruz)。
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