爱华网Stripping buffer 的配方
两种方法，国际常用，符合国际标准。
Membrane Stripping Two protocols are presentedbelow. The first is applicable to any chemiluminescent substratesystem and uses a combination of detergent and heat to release theantibodies. The second is commonly used for applications whereantibodies have to be separated from an antigen and employs low pHto alter the structure of the antibody in such a way that thebinding site is no longer active.
Neither method will remove the colored precipitates generated fromchromogenic detection systems (e.g., BCIP, 4CN, DAB and TM.However, it is still possible to analyze the blot with anotherantibody specific to a different target protein.
Important: The blot should not be allowed to dry between rounds ofimmunodetection. Any residual antibody molecules will bindpermanently to the membrane if it is allowed to dry.
Protocol 1. Stripping by Heat and Detergent
Applicable to any chemiluminescent substrate system.
Required Equipment and Solutions
Stripping solution: 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mMTris-HCl, pH 6.7
Phosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2,0.9% (w/v) NaCl
Shallow tray, large enough to hold the membrane
Procedure
1. In a fume hood, place the blot in stripping solution and agitatefor 30 minutes at 50 °C.
2. Place the blot in buffer and agitate for 10 minutes. Repeat withfresh buffer.
3. (Optional) Repeat the initial detection protocol (omitting theprimary antibody step) to make sure that the antibodies have beeninactivated or stripped from the membrane.
4. Place the blot in buffer and agitate for 10 minutes.
5. Proceed to the blocking step for the next round ofdetection.
Protocol 2. Stripping by Acidic pH
Applicable to any chemiluminescent substrate system.
Required Equipment and Solutions
Stripping solution: 25 mM glycine-HCl, pH 2, 1% (w/v) SDS
Phosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2,0.9% (w/v) NaCl
Shallow tray, large enough to hold the membrane
Procedure
1. Place the blot in stripping solution and agitate for 30minutes.
2. Place the blot in buffer and agitate for 10 minutes. Repeat withfresh buffer.
3. Proceed to the blocking step for the next round ofdetection.
还有自己整理的
METHOD1
1、stripping buffer: 62.5mmol/l Tris PH6.7；100mmol/lbeta-mercaptoethanol；2%SDS
二次发光protocol:1.stripping buffer洗膜：50度水浴30分钟，摇床上摇10-20分钟。
2、1*PBST洗：摇床上摇30分钟。
3、封闭，加一抗，二抗（同第一次发光）
METHOD2
（50ml总量）：β-mercaptoethanol 342 μl；20% SDS 5 ml；1M/L Tris-Cl pH 6.73.125 ml；加ddH2O至 50 ml。
方法：将用过的膜浸入strippingbuffer中，置50℃水浴箱中30min，间断振摇。之后用TTBS洗3*5min就好。此时你已经可以按新的转移好的膜来再次使用了。
该方法的优点：省事，省力，省钱，符合国际惯例。
METHOD3
1、beta-metaptoethanol 35ul
2、10%SDS 1ml
3、tris (0.5M，pH6.7) 625ul
4、dH2O 3.34ml
50-55℃，30min。
这是LumiGLO 说明书带的protocol
Stripping and Reprobing a Western blot:
1. Remove membrane from plastic following initial detection withLumiGLO.
2. Rinse membrane for 30 - 90 minutes at 70°C in 2% SDS(w/v)/62.5mM Tris-HCl (pH 6.8 at 20°C)/100 mMβ-mercaptoethanol.
3. Wash membrane 2 times in 10 mM Tris-HCl (pH 7.4 at 20°C)/150 mMNaCl.
4. Block membrane for 2.5 hours with Detector Block or 10 mMTris-HCl (pH 7.4 at 20°C)/150 mM NaCl/5% nonfat dry milk.
5. Repeat detection procedure.
一般来说为了节约样品、试剂，同时更重要的是尽量使对照组之间的调控关系不因样品的差异而变化，我们在做WB的时候免不了要重复使用同一张膜。刚才回帖的时候谈到了StrippingBuffer，觉得有些经验值得专门贴出来。
***NC膜我用的少，以下经验主要对PVDF膜而言。
关于Stripping Buffer：现在很多公司都推出了不同的StrippingBuffer，还有很多ECL试剂盒也会告诉你Stripping的方法。我只用过sigma的，效果不如他吹嘘的那么好。其实很多实验室口口相传的StrippingBuffer才是真正好的方法。我用的比较好的配方是：
100 mM ß-mercapto-ethanol, 2% SDS, 62.5 mM Tris-Cl pH 6.8
50mlbuffer当中：
ß-mercaptoethanol 342 µl
20% SDS 5 ml
Tris-Cl pH6.7 3.125 ml
add H2O to 50 ml
65C X 30min （效果不好的话用45min-1hour）
TBST洗10min X 3次（别嫌麻烦）
blocking in BSA or milk 1hour（别嫌麻烦）
reuse
***
-Stripping的关键是SDS浓度，如果你觉得洗的不理想的话可以适当增加SDS（每次0.2%的递增）。洗过了也可以降低一点。
- ß-mercaptoethanol我从300ul-1ml都用过，没觉得影响太大。这东西还是有毒的，味道不好闻，尽量在通风橱里面操作。
- 温度：50-65C都可以，不过65C的洗脱确实好一些。
- Stripping之后一定要洗干净，不然一抗与蛋白的结合有问题
- 如果膜不急用的话，TBST洗10min X 3次之后可以用 1x TBS（NOTween20）泡着，最好是用塑料袋封在TBS里面，低温（4℃保持）。一个月是没问题的。
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