StrippingBuffer配方

1、Stripping Buffer 配方及說(shuō)明兩種方法，國(guó)際常用，符合國(guó)際標(biāo)準(zhǔn)。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from an antigen and employs low pH to alter the structure of the antibody in such a way that the binding site is no longer active.Neither method will remove the colored precipitates generated from chromogenic detection systems (e.g., BCIP, 4CN, DAB and TM.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 分鐘，搖床上搖 10-20 分鐘。2、IxPBST洗：搖床上搖30分鐘。3 、封閉，加一抗，二抗（同第一次發(fā)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：將用過(guò)的膜浸入 stripping buffer中，置50 C水浴箱中30min，間斷振搖。用 TTBS 洗 3*5min 就好。此時(shí)你已經(jīng)可以按新的轉(zhuǎn)移好的膜來(lái)再次使用了。 該方法的優(yōu)點(diǎn)：省事，省力，省錢(qián)，符合國(guó)際慣例。METHOD31 、 beta-metaptoethanol 35ul2、10%SDS 1ml3、tris （0.5M ， pH6.7） 625ul4、dH2O 3.34ml方法：50-55C， 30min。

10、以下經(jīng)驗(yàn)主要對(duì) PVDF 膜而言。關(guān)于 Stripping Buffer ：現(xiàn)在很多公司都推出了不同的 Stripping Buffer ，還有 很多 ECL 試劑盒也會(huì)告訴你 Stripping 的方法。我只用過(guò) sigma 的，效果不如 他吹噓的那么好。其實(shí)很多實(shí)驗(yàn)室口口相傳的 Stripping Buffer 才是真正好的 方法。65C ， 30min （效果不好的話(huà)用 45min-1hour ）TBST 洗 10min X 3 次（別嫌麻煩） blocking in BSA or milk 1hour （別嫌麻煩） reuseStripping 的關(guān)鍵是 SDS 濃度，如果你覺(jué)得洗的不理想的話(huà)可以適當(dāng)增加 SDS （每次0.2%的遞增），洗過(guò)了也可以降低一點(diǎn)。?-mercaptoethanol 我從 300ul-1ml 都用過(guò)，沒(méi)覺(jué)得影響太大。這東西還是 有毒的，味道不好聞，盡量在