Stripping Buffer 配方及说明
Membrane Stripping Two protocols are presented below. The first is applicable to any chemiluminescent substrate system and uses a combination of detergent and heat to release the antibodies. The second is commonly used for applications where antibodies have to be separated from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active.
Neither method will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TM. However, it is still possible to analyze the blot with another antibody specific to a different target protein.
Important: The blot should not be allowed to dry between rounds of immunodetection. Any residual antibody molecules will bind permanently to the membrane if it is allowed to dry.
火锅餐具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 mM Tris-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 agitate for 30 minutes at 50 °C.
2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
3. (Optional) Repeat the initial detection protocol (omitting the primary antibody step) to make sure that the antibodies have been inactivated 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 of detection.
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 30 minutes.
2. Place the blot in buffer and agitate for 10 minutes. Repeat with fresh buffer.
3. Proceed to the blocking step for the next round of detection.
整理的方法
闪光灯柔光罩METHOD1
stripping buffer:
62.5mmol/l Tris PH6.7；100mmol/l beta-mercaptoethanol；2%SDS
protocol:
1.stripping buffer洗膜：50度水浴30分钟，摇床上摇10-20分钟。
2、1ⅹPBST洗：摇床上摇30分钟。
3、封闭，加一抗，二抗（同第一次发光）
METHOD2
β-mercaptoethanol 342 μl；20% SDS 5 ml；1M/L Tris-Cl pH 6.7 3.125 ml；加ddH2O至 50 ml。
方法：
将用过的膜浸入硅铁合金
stripping buffer中，置heff50℃水浴箱中30min，间断振摇。 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
10.10.0.10
隐私保护通话4、dH2O 3.34ml
方法：
50-55℃，30min。
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Stripping Buffer，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Stripping Buffer才是真正好的方法。
65℃ ， 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（NO Tween20）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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