StrippingBuffer配方

1、Stripping Buffer 配方及说明两种方法，国际常用，符合国际标准。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

2、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from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active.Neither method will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TM.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

3、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.Protocol 1. Stripping by Heat and Deterge

4、nt Applicable to any chemiluminescent substrate system.Required Equipment and SolutionsStripping solution: 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mM Tris-HCl, pH 6.7Phosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaClShallow tray, large enough to hold the membranePr

5、ocedure1. 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 anti

6、bodies 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 SolutionsStr

7、ipping solution: 25 mM glycine-HCl, pH 2, 1% (w/v) SDSPhosphate buffered saline (PBS): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaClShallow tray, large enough to hold the membraneProcedure1. Place the blot in stripping solution and agitate for 30 minutes.2. Place the blot in buffer and agitate for

8、 10 minutes. Repeat with fresh buffer.3. Proceed to the blocking step for the next round of detection.整理的方法METHOD1stripping buffer:62.5mmol/l Tris PH6.7 ； 100mmol/l beta-mercaptoethanol ； 2%SDSprotocol:1. stripping buffer 洗膜： 50 度水浴 30 分钟，摇床上摇 10-20 分钟。2、IxPBST洗：摇床上摇30分钟。3 、封闭，加一抗，二抗（同第一次发光）METHOD2m

9、ercaptoethanol 342； 20% SDS 5 ml ； 1M/L Tris-CI pH 6.7 3.125 ml ；力廿ddH2O 至 50 ml 。方法：将用过的膜浸入 stripping buffer中，置50 C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。METHOD31 、 beta-metaptoethanol 35ul2、10%SDS 1ml3、tris （0.5M ， pH6.7） 625ul4、dH2O 3.34ml方法：50-55C， 30min。

10、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。65C ， 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） reuseStripping 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。?-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