15）关闭电源并撤去连接的导线，弃去电泳缓冲液。连同上槽一起将凝胶夹层取出。
16）将凝胶定位以便识别加样的顺序，将凝胶板从上槽解离出来，放在一叠吸水纸或纸巾上。
17）小心将封边的垫片抽出一半，并以此为杠杆橇起上面的玻璃平板，使凝胶暴露出来。
18）小心从下面的玻璃平板上移出凝胶，在凝胶的一角切去一小块以便在染色及干胶后仍能认出加样次序，接着就可进行蛋白质的检测。
19)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 recommendations
20)、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！）
21)、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℃.
22)、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.
23)、清洗
将硝酸纤维素膜放入1X TBST中清洗3次，每次5分钟。
24)、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。
25)、清洗
同22，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。
26)、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
