（五）免疫反應
（1）將膜用TBS從下向上浸濕后，移至含有封閉液的平皿中，室溫下脫色搖床上搖動封閉1h。
（2）將一抗用TBST稀釋至適當濃度（在1.5ml離心管中）；撕下適當大小的一塊兒保鮮膜鋪于實驗臺面上，四角用水浸濕以使保鮮膜保持平整；將抗體溶液加到保鮮膜上；從封閉液中取出膜，用濾紙吸去殘留液后，將膜蛋白面朝下放于抗體液面上，掀動膜四角以趕出殘留氣泡；室溫下孵育1～2h后，用TBST在室溫下脫色搖床上洗兩次，每次10min；再用TBS洗一次，10min。
（3） 同上方法準備二抗稀釋液并與膜接觸，室溫下孵育1～2h后，用TBST在室溫下脫色搖床上洗兩次，每次10min；再用TBS洗一次，10min，進行化學發光反應。
（六）化學發光，顯影，定影
（1）將A和B兩種試劑在保鮮膜上等體積混合；1min后，將膜蛋白面朝下與此混合液充分接觸；1min后，將膜移至另一保鮮膜上，去盡殘液，包好，放入X-光片夾中。
（2）在暗室中，將1×顯影液和定影液分別到入塑料盤中；在紅燈下取出X－光片，用切紙刀剪裁適當大小（比膜的長和寬均需大1cm）；打開X-光片夾，把X－光片放在膜上，一旦放上，便不能移動，關上X-光片夾，開始計時；根據信號的強弱適當調整曝光時間，一般為1min或5min，也可選擇不同時間多次壓片，以達最佳效果；曝光完成后，打開X-光片夾，取出X-光片，迅速浸入顯影液中顯影，待出現明顯條帶后，即刻終止顯影。顯影時間一般為1～2min（20～25℃），溫度過低時（低于
16℃）需適當延長顯影時間；顯影結束后，馬上把X-光片浸入定影液中，定影時間一般為5～10min，以膠片透明為止；用自來水沖去殘留的定影液后，室溫下晾干。
應注意的是：顯影和定影需移動膠片時，盡量拿膠片一角，手指甲不要劃傷膠片，否則會對結果產生影響。
（七） 凝膠圖象分析
將膠片進行掃描或拍照，用凝膠圖象處理系統分析目標帶的分子量和凈光密度值。
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蛋白表達通過 Western blot進行檢測。已達融合生長的單層細胞用冷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離心去除細胞碎片后，取50?g樣品與上樣緩沖液混合，煮沸5min，進行10% SDS-PAGE電泳，轉硝酸纖維素膜(Pall
Corporation)。將膜在含5%脫脂奶粉的TBST(10mM Tris-HCl, pH7.5,150mM NaCl,
0.05%Tween-20)中室溫下封閉1h，隨后加入鼠抗人ezrin一抗(Maixin-Bio)，抗鼠IgG-HRP二抗，ECL化學發光試劑檢測(Santa
Cruz)。