Western Blot 实验计划完整版.doc

Western Blot 实验计划完整版一、SDS-PAGE1、分离胶的配制10% 10%30:0.8丙烯酰胺/双丙烯酰胺 2.67 mL 4.005mL去离子水 3.33 mL 4.995mL分离胶缓冲液(4X) 2.00 mL 3.00mL 8.00 mL 12.00mL10%(w/v)APS(现用现配) 45μL 67.5μLTEMED 12μL 18μL2、压缩胶的配制3%30:0.8丙烯酰胺/双丙烯酰胺 0.75 mL去离子水 3.00 mL压缩胶缓冲液(4X) 1.25 mL 5.00 mL10%(w/v)APS(现用现配) 30μLTEMED 8μL先制备蛋白样品，后灌注压缩胶，压缩胶灌注后应在20~40min内使用。

3、蛋白样品的制备蛋白样品 8μL上样缓冲液(loading buffer/laemmili buffer) 12μL煮沸5min冷却至室温短暂离心之后可加样，加样孔的深度至少为5~8mm拔出梳子后，如加样孔有气泡，可向其中加入适量电泳缓冲液将气泡驱除从烧水开始，蛋白样品的制备大约需要35min4、电泳压缩胶电压：80V（10mA）分离胶电压：120V（20mA）当溴酚兰指示剂到达凝胶底部时，即停止电泳5、染色考马斯亮蓝染色液35min，摇床摇动（为获得最大分辨率，5%凝胶染色2小时，10%凝胶染色4小时）用清水反复漂洗几次高甲醇脱色液25min，摇床摇动（也可用7%（v/v）冰乙酸）低甲醇脱色液，摇床摇动二、Western blot1、润湿准备6张Whatman 3#滤纸、一张硝酸纤维素膜，尺寸与凝胶大小相仿，先放入水中3分钟，放入转膜缓冲液中5分钟2、起胶将凝胶从电泳槽中取出，放入转膜缓冲液中10分钟3、三明治的制作按以下顺序放置：3层Whatman 3#滤纸、凝胶、硝酸纤维素膜、3层Whatman 3#滤纸切记：每层之间用滴管将其中的气泡全部赶出，决不允许气泡存在。

4、海绵的润湿用转膜缓冲液将海绵润湿5、转膜将凝胶面与负极相连，硝酸纤维素膜与正极相连100V，1小时）要放于4℃1. For transfer of proteins smaller than 20 kDa, transfer proteins from gel to PVDF (polyvinylidene difluoride) membrane at 1Amp constant current for 45 mins or equivalent (250mAmp for 3 hours or 500mAmp for 90 minutes) in transfer buffer (25mM Tris, 190mM glycine, 20% MeOH). 2. For transfer of proteins smaller than 120 kDa, transfer proteins from gel to PVDF membrane at 1Amp constant current for 1 hour or equivalent (250mAmp for 4 hours or 500mAmp for 2 hours) in transfer buffer (25mM Tris, 190mM glycine, 20% MeOH). 3. For proteins larger than 120kDa, transfer to PVDF membrane at 1Amp constant current for 90 minutes or equivalent (250mAmp for 6 hours or 500mAmp for 3 hours) in transfer buffer + SDS (25mM Tris, 190mM glycine, 20% MeOH, 0.05% SDS). 4. For Proteins larger than 250kDa, transfer to PVDF membrane at 1Amp constant current for 1 hour and 45 minutes or equivalent (500mAmp for 3.5 hours) in transfer buffer + SDS (25mM Tris, 190mM glycine, 20% MeOH, 0.05% SDS). Add transfer (or CAPS) buffer to the transfer unit according to manufacturer1s directions. Transfer over night with a setting of 20V / 40 mA or for 3 hours at 70V/160 mA. The transfer process needs to take place under cold temperatures to prevent the gel from sticking to the membrane. This can be accomplished either by using a water cooling core in the transfer unit or placing the entire unit at 4. Please follow the manufacturer1s recommendations6、清洗将硝酸纤维素膜放入1X TBST中清洗3次，每次5分钟。

摇床摇动这步忘了！）7、封闭1. Remove the blot from the transfer apparatus or staining tray and immediately place into blocking buffer (1% BSA, 10mM Tris pH 7.5, 100mM NaCl, 0.1% Tween 20). 2. Incubate the blot for 1 hour at 37℃, 2 hours at room temperature, or overnight at 4℃.8、一抗孵育一抗用TBST1:1000稀释，将硝酸纤维素膜放入其中，37℃孵育1.5小时，(或4℃过夜)摇床摇动Probe with primary antibody in TTBS/1% NFDM for 1 hr. at room temperature. Primary antibody should be diluted as specified on product data sheets. If these values are not available, use the following guidelines for initial experiments: apply antisera or ascites at 1:500 to 1:5,000, apply purified primary antibodies at a concentration of 1 ug/ml.9、清洗将硝酸纤维素膜放入1X TBST中清洗3次，每次5分钟。

10、二抗孵育二抗用TBST1:8000稀释，将硝酸纤维素膜放入其中，37℃孵育1小时，摇床摇动11、清洗同13，之后，用1X TBS在清洗2次，每次5分钟，以洗去膜上的Tween 20，因为它可以阻碍底物的沉积12、显色按如下配方配制显色液：1mL水+1滴（大约50微升）试剂A(之后混匀)+1滴试剂B+1滴试剂C将硝酸纤维素膜放入其中孵育，一旦显色，立即放入去离子水中终止反应Considerations:· Perhaps the most common problem in western blotting is the occurrence of background staining. The best remedy for high background (in many cases) is simply to dilute the primary antibody further. Other solutions include ensuring that detergent is used in the blocking reagent, using an alternate blocking reagent (casamino acids, BSA, serum), and decreasing the amount of protein applied to the electrophoresis gel.· Occurrences of extra bands in the blot can be resolved by several strategies. Run a control blot omitting the primary antibody to determine if the secondary antibody is the source of the problem. Replacing the secondary antibody with a different lot or a similar reagent from a different source can provide resolution. Spurious bands below the targeted molecular weight suggest that the protein is being degraded in the experiment; inclusion of protease inhibitors can help.· No or low signal can be remedied by loading more protein in the gel or increasing the amount of primary and/or secondary antibody applied to the blot.· Some antibodies will not bind in the presence of detergent; consult the datasheet for each antibody prior to performing any procedure. 本实验所需全部试剂的配方：SDS-PAGE一、丙烯酰胺/双丙烯酰胺储液（30:0.8）1000mL30%(w/v) 丙烯酰胺 300g0.8%(w/v) 双。