Do you have a recommended protocol for stripping/re-probing blots?

The western blot stripping/re-probing protocol for our Western Blot Stripping Buffer (5X) #91925 is below. However, it is important to note that we typically advise against stripping/re-probing because stripping can be incomplete and/or lead to inconsistent results through the loss of target proteins from the membrane. If possible, it is much better to run fresh immunoblots with each individual antibody on duplicated sets of samples.

A. Solutions and Reagents
注：利用反渗透去离子水 (RODI) 或同等级别的水制备溶液。

提供的试剂：
1. Western Blot Stripping Buffer (5X) #91925: To prepare 20mL of 1X Stripping Buffer, combine 4mL 5X Stripping buffer with 140uL 2-mercaptoethanol (0.1M final concentration) and 15.86mL RODI water.

其他试剂（未提供）：
1. 10X Tris Buffered Saline with Tween® 20 (TBST) #9997: To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
2. 2-mercaptoethanol

B. 洗脱膜
注： Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different-sized membranes, adjust volumes accordingly.
注：This protocol begins after membrane exposure to film or digital imager. The best results are obtained if the membrane is not allowed to dry. Membranes can be stored in 1X TBST at 4°C.
注： Quantitative comparison of targets before and after stripping is not recommended due to the removal of small amounts of membrane-bound protein with each round of stripping.
1. 在室温下用 15 ml 1X TBST 洗涤 3 次，每次 5 分钟。
2. Incubate membrane in 20 ml of Stripping Buffer for 45 minutes to
1 hr at 50°C with slight agitation, if available. 根据抗体信号强度，可能需要优化孵育时间和温度。The incubation time can be adjusted up to 2 hours while the incubation temperature can be adjusted to 70°C if needed.
3. 在室温下用 15 ml TBST 洗涤五次，每次 5 分钟。
4. （可选）要检查洗脱效率并确认原始信号已被去除，请将膜与适当的二抗一起孵育，然后使用推荐的检测试剂进行蛋白质检测。如果洗脱完成，继续在室温下用 15 ml TBST 洗涤膜五次，每次 5 分钟。如果洗脱不充分，重复步骤 2 到 4。
5. 该膜已准备好重复使用。在添加一抗之前继续进行封闭步骤。