Western抗体洗脱液

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

注意事项：
1. 膜再生液实质是在不影响膜上结合的抗原的条件下将抗体洗脱下来，实际操作中，膜类型、抗体类型、浓度及抗原特性都影响洗脱难易度。
2. 适用于 ECL 和类似的化学发光试剂进行的 Western 检测。不适用于非化学发光试剂进行的 Western检测，例如 DAB，NBT/BCIP。
3. 用 ECL 显色后，再生液洗脱几分钟后，可再次加入 ECL 显色液显影，如胶片上显示条带，表明抗体未除净，须进一步在再生液中浸泡，至胶片上无显色。
4. 使用脱脂奶粉封闭的膜要比使用 BSA 封闭的膜更容易再生。
5. 尽量避免让膜干燥。

在遇到一抗二抗去除效果欠佳时，可以考虑适当延长一抗二抗去除液的孵育时间，同时确保孵育时的温度不低于20℃。温度过低时一抗二抗的去除效果会下降。同时也可以考虑尝试其它的一抗二抗去除液，以摸索出的适合您实验条件的Western一抗二抗去除液。