1、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 3.00mL8.00 mL 12.00mL10%(w/v)APS(现用现配) 45L 67.5LTEMED 12L 18 L2、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 mL5.00 mL10%(w/v)APS(现用现配) 30LTEMED 8L先制备蛋白样品，后灌注压缩胶，压缩胶灌
2、注后应在 2040min 内使用。3、蛋白样品的制备蛋白样品 8L上样缓冲液(loading buffer/laemmili buffer) 12L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 58mm。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 25m
3、in，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。
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 prot
5、eins 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 constan
6、t 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.
7、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
8、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
9、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
10、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
11、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。Consider
12、ations: 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 altern
13、ate 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 sou
14、rce 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. N
15、o 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：SD
16、S-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) 双丙烯酰胺 8g加去离子水，至终体积 1000mL。经 0.22m 膜过滤。存于 4暗处。二、分离胶缓冲液（4X）1.5mol/L Tris-HCL(pH8.0)0.4%(w/v)SDS500mL 1000mLTris-Base 90.8g 181.7g10%(w/v) SDS 20mL 40mL加去离子水，至所需终体积。室温下用 HCL 调 pH 至 8.0。经 0.22m 过滤。存于 4。三、压缩胶缓冲液（4X）0.5mol/L Tris-HCL(pH6.8)0.4%
17、(w/v)SDS500mL 1000mLTris-Base 30.3g 60.6g10%(w/v) SDS 20mL 40mL加去离子水，至所需终体积。室温下用 HCL 调 pH 至 6.8。经 0.22m 过滤。存于 4。四、电机缓冲液（4X）0.1mol/L Tris0.768mol/L 甘氨酸4000mL 8000mLTris-Base 48.8g 96.9g甘氨酸 230.6g 461.2g加去离子水，至所需终体积。不必调 pH。此比例时 Tris 碱-甘氨酸的 pH 应为8.3。室温下存于大容器中。五、SDS(10%;w/v)100mL取 10gSDS 溶于去离子水中，至终体积 10
18、0mL。室温下存于洁净瓶中。六、电泳缓冲液（1X）1/4 体积的 4X 电泳缓冲液与 3/4 体积的去离子水混合。然后加 1/100 体积的10%SDS 至终浓度 0.1%(w/v)SDS。实例如下：800mL电极缓冲液 200mL去离子水 592mL10%(w/v)SDS 8mL800mL七、样品缓冲液(2X) 储液0.25mol/LTris-HCL(pH6.8)4%(w/v)SDS20%(w/v)甘油痕量溴酚兰配制此溶液时，应极小心，戴手套，并避免角蛋白（皮肤上即存在此蛋白）对缓冲液的污染。100mL压缩胶缓冲液（4X） 50mLSDS(固体干粉) 4g甘油 20mL溴酚兰（溶液呈深蓝色）
19、 2mg加去离子水至 100mL 终体积。此缓冲液可室温下保存或等分冻干保存，但用前必须温热以确保 SDS 于溶液中。应保证反复使用此溶液的同一储液而不被污染。等分保存将有助于避免这个问题。八、含 2-巯基乙醇的样品缓冲液（1X）用前配制样品缓冲液的工作液，稀释 2X 样品缓冲液，加入 2-巯基乙醇至终浓度为 5%（v/v）。例如：2X 样品缓冲液 100L去离子水 90L2-巯基乙醇 10L200L九、过硫酸铵（APS）(10%;w/v)取 3g 过硫酸铵，溶于去离子水，至终体积为 30mL。此溶液 4下在短暂的数日内是稳定的，但建议，对每一组新胶均应新鲜配制。十、考马斯亮蓝染色液（1000
20、mL）考马斯亮蓝 R250 2.5g甲醇 454mL冰醋酸 92mL去离子水 454mL经 Whatman #1 滤纸过滤。十一、高甲醇脱色液1000mL 2000mL甲醇 454mL 908mL冰醋酸 75mL 150mL去离子水 471mL 942mL十二、标准（低甲醇）脱色液1000mL 2000mL甲醇 50mL 100mL冰醋酸 75mL 150mL去离子水 875mL 1750mLWestern Blot一、膜转移缓冲液（Transfer buffer）组分浓度：39mM Glycine, 48mM Tris, 0.037%(w/v)SDS, 20%(v/v)甲醇配制量：1L配制方
21、法：1、称量下列试剂，置于 1L 烧杯中。Glycine 2.9gTris 5.8gSDS 0.37g2、向烧杯中加入约 600mL 的去离子水，充分搅拌溶解。3、加去离子水将溶液定容至 800mL 后，加入 200mL 的甲醇。4、室温保存。二、TBS(5X)组分浓度：100mM Tris-Base, 0.685M NaCl配制量：500mL配制方法：1、称量下列试剂，置于 500mL 烧杯中。Tris-Base 0.6075gNaCl 20.0157g2、向烧杯中加入约 400mL 的去离子水，充分搅拌溶解。3、调节 pH 至 7.64、加去离子水将溶液定容至 500mL 后，4保存。三、TBST(Washing buffer)100mL 5X TBS0.25mL Tween 20Q.S.to 500mL四、Blocking buffer(1%BSA in TBST)20mL 5X TBS1g BSAQ.S.to 100mL
