Hi, The membrane should be washed off the ECL reagent using PBS for 5-10' and then stored in the PBS at 4degrees or even without PBS at room temperature. The efficiency of getting a good signal decreases with time and also the number of times the membrane is stripped. However, for a loading control like B tubulin the signal should be very strong and you should be able to see a strong signal even after multiple strippings. The stripping buffer is Tris based pH 6.8 and has BME and SDS and should be warmed up t0 56degrees and the membrane can be stripped in a 56degree water bath for 30' with intermittent shaking. Then the membrane should be rinsed multiple times with PBST/TBST (10' washes at least 4-5times) until the smell of BME is gone. Then it can be blocked and primary, wash, secondary, wash and develop. Hope it helps. Good luck!

Also, some of the loading controls tend to have very strong signal, so there might not be a need to strip the blot after the first ECL developing. If the bands are sufficiently far away from each other, just wash off the ECL reagent with PBS and add a new primary and secondary antibody. I'd say the less manipulation with the membrane, the better.

Thank you so much Bharat and Adam.

I wanted to store the blot for two days before stripping it,so was a little concerned whether it would affect the signal.

The storing of your membrane depend of its chemistry. With nitrocellulose, you could keep it at 4°C. For PVDF, you could dry it on a wattman paper and freeze it at -20°C. To use it, you need to soak it in ethanol followed by 10min in water. After, you need to perform the blocking step.

And I agree with Adam, no striping is better.

Hi, for the stripping procedure you have well answered.

If you need a mild stripping (you have to remove a weak signal) I also used with good results:

0,1M Glycine

20 mM Magnesium acetate (4H2O)

50 mM KCl pH 2,2

Incubate two times with this buffet for 10 min with gentle agitation

Wash with TBST or PBST 3x 5min

Block with milk (or BSA)

For the membrane storage if you preserve well the membrane you can use the membrane for antibody incubation or stripping after months. Someone store used wet storage other dry storage, I used both always at 4degrees.

Two days in PBS / 4 °C is OK (for nitrocellulose membrane, that is). I have no experience with drying the membrane after developing it.

Better possibility, I think, would be to dry and store it right after blotting (in RT or 4 °C, both works) and then do the developing two days in row.

Thanks so much everyone

If you trying to detect a loading control, you probably will not need to strip your membrane. If your tubulin antibody is very good, you can incubate it at the normal or slightly increased concentration, allowing you to detect your band of interest over signals you previously detected. If your tubulin is already conjugated, then you will not need any secondary antibody, thus reducing the possibility of detecting previous signal.