Test for complete removal of secondary antibodies: After stripping, incubate the membrane with fresh Western Blot reagents(such as ECL) and expose to X-film. If no signal is detected for 5-min exposure, the secondary antibodies have been successfully removed. Test for complete removal of the primary antibodies: Incubate the membrane with HRP-conjugated secondary antibodies, followed by 3x washing. Incubate with fresh Western Blot reagents(such as ECL) and expose to X-film. If no signal is detected for 5-min exposure, the primary antibodies have been successfully removed.

Yes. OneMinute® stripping buffer efficiently removes antibodies from both nitrocellulose and PVDF membranes.

Re-blocking the membrane is usually not necessary after stripping with OneMinute®, but it is recommended to re-probe the membrane with antibodies in the buffer containing 1% milk. However, re-blocking may help to decrease background in some situations.

No, but the luminescent signals are quenched.  HRP or AP conjugated to secondary antibody cleaves a chemiluminescent substrate, and the reaction product produces luminescent signal and precipitates on the membrane. OneMinute® strips off primary/secondary antibodies and quenches the luminescent signals, but cannot remove the precipitate.

OneMinute® Stripping buffer is harsh for primary and secondary antibodies, however it is very soft for antigens bound on the membrane. In one of our tests, 50% antigens were still remained on the membrane after 10-time stripping (Figure 6). However, the situation is different from case to case, it depends both on the stability of antigens on membrane and the sensitivity of antibodies. The antigens may withstand stripping as many as 10 times or as few as one time.

OneMinute® can not break strong interactions, such as avidin-biotin interaction. In some rare case, OneMinute® does not work very well for specific antibodies. OneMinute® does not strip off fluorescent signals.

Yes. OneMinute® Stripping buffer expires in several months. OneMinute®Plus has much longer shelf life (~18 months).  Please find the expiration date on the bottle.

No. In chemifluorescent method, non-fluorescent substrates are enzymatically converted into fluorescent products by HRP or AP conjugated to secondary antibody. The fluorescent products precipitate on the membrane and are detected by scanner or CCD camera. OneMinute® strips off primary and secondary antibodies, but cannot remove or quench the fluorescent precipitates.

No. Unacceptable fluorescent background could be generated although the antibodies can be stripped off. OneMinute® is designed for procedures using chemiluminescent substrates.

Yes. Keep in mind to expose your membrane to X-film for the signals detection. OneMinute® works very well in places where X-film is used to detect signals in subsequent re-blotting.