Stripping is the term used to describe the removal of primary and secondary antibodies from a western blot membrane. Stripping is useful when one wants to investigate more than one protein on the same blot, for instance a protein of interest and a loading control. When probing for multiple targets, stripping and re-probing a single membrane instead of running and blotting multiple gels has the advantage of saving samples, materials, and time.

It is not advisable to make quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane. For the same reason, a stripped membrane should not be probed to demonstrate the absence of a protein.

A PVDF membrane is highly recommended to minimize loss of sample protein. Note also that colorimetric/chromogenic detection reagents will leave a permanent visible stain on the membrane that can interfere with subsequent detection of targets of similar molecular weights. Chemiluminescent reagents such as ECL are recommended as they will not leave a stain and are more sensitive than colorimetric reagents.

Procedure

1.Warm the buffer to 50°C
2.Add the buffer to a small plastic box which has a tight lid; use a volume that will cover the membrane
3.Add the membrane. Incubate at 50°C for up to 45 min with some agitation
4.Dispose of the solution as required for ß-mercaptoethanol based buffers
5.Rinse the membrane under running water tap for 1–2 min
6.Traces of ß-mercaptoethanol will damage the antibodies. Wash extensively for 5 min in TBST
7.Ready for blocking

注意事項(xiàng)：
1. 膜再生液實(shí)質(zhì)是在不影響膜上結(jié)合的抗原的條件下將抗體洗脫下來，實(shí)際操作中，膜類型、抗體類型、濃度及抗原特性都影響洗脫難易度。
2. 適用于 ECL 和類似的化學(xué)發(fā)光試劑進(jìn)行的 Western 檢測。不適用于非化學(xué)發(fā)光試劑進(jìn)行的 Western檢測，例如 DAB，NBT/BCIP。
3. 用 ECL 顯色后，再生液洗脫幾分鐘后，可再次加入 ECL 顯色液顯影，如膠片上顯示條帶，表明抗體未除凈，須進(jìn)一步在再生液中浸泡，至膠片上無顯色。
4. 使用脫脂奶粉封閉的膜要比使用 BSA 封閉的膜更容易再生。
5. 盡量避免讓膜干燥。

在遇到一抗二抗去除效果欠佳時(shí)，可以考慮適當(dāng)延長一抗二抗去除液的孵育時(shí)間，同時(shí)確保孵育時(shí)的溫度不低于20℃。溫度過低時(shí)一抗二抗的去除效果會(huì)下降。同時(shí)也可以考慮嘗試其它的一抗二抗去除液，以摸索出的適合您實(shí)驗(yàn)條件的Western一抗二抗去除液。