No signal on dried PVDF membranes after MeOH rehydration/stripping/reprobing - (Sep/22/2010 )

Have been trying to reprobe dried PVDF WB membranes for loading controls, or input controls. I can never get a signal. Not even background. Blank film even with O/N exposure every single time.

I rehydrate in MeOH briefly (10 seconds), then rinse with water, then wash in TBST several times. I then strip with Pierce Western Restore stripping buffer for just 5 minutes, wash extensively in TBST, reblock, apply primary Ab O/N, and then wash and apply secondary, etc.

I would think this just didn't work for dried membranes, but recently I gave an old, dried membrane (stored at RT, wrapped in plastic wrap in my lab notebook for several months, just like the others) to a colleague in another lab that had been probed with a crappy antibody and was pure black upon development. I considered it unusable, but he came back and told me he had stripped it and reprobed it 8 times successfully. He said he used the Pierce Western Restore stripping buffer.

Most recently I tried this on a WB on IP samples. The IP pulled down binding partners, but I had neglected to do an input control at that time, so I was attempting to probe for the protein I pulled down. This is a highly abundant protein, and I have evidence (from identical samples run on other gels) that the IP worked beautifully. However, I want a control to show for *this* western and I can't get it. Repeating this experiment would not be trivial, and I can still see the molecular weight markers on the membrane, so I do not think I stripped all the protein off. Unless, perhaps, there is some reason why successive treatment with methanol, water, TBST, and stripping buffer liberates all traces of protein from the membrane.

Am I missing something here? I've tried storing membranes in the fridge in TBST overnight and reprobing (without rewetting or stripping) and also can't get any signal from them when reprobed. I know all of our WB reagents work, since the fresh WBs work perfectly.

I would greatly appreciate *ANY* insight anyone could offer. Thanks.

So you're saying that after storing a freshly visualized blot overnight in TBST, if you reprobe with the same antibody mix the next day and visualize again, you get no signal at all?

If I store overnight in TBST, I'll get signal. I can reprobe maybe 1 or 2 times, then all signal is gone even if I loaded 100 ug of protein. If I store in TBST longer, say, for a week, I'll have probems with signal.

I'm wondering if residual stripping buffer is interfering with the chemiluminescence reaction... I wash quite extensively, though. I *really* need to get signal from this IP western blot and I have no idea why this didn't work.

Apparently, there are two formulations of Restore Western Blot Stripping Buffer – the "original gentle formulation" and another "stronger formulation for tenacious antibodies" (see here). Perhaps the "crappy antibody" you used on the western you gave to your colleague is effectively removed by the original formulation, but the antibodies you used in the westerns you can't get signal from after stripping are not.

Call Pierce for tips -- higher incubation temperature, longer incubation times, etc. -- or try the Restore Plus Western Blot Stripping Buffer.