There are a couple things to consider. The first is that proteins naturally leech out of the membrane over time. This can be slowed down but not prevented so you will notice your signal dropping over time.

I think your main issue is storing the membrane with PBS + Tween for a long duration. Tweens job is to strip protein off the membrane preventing non-specific interactions but leaving it on the membrane for multiple days could be stripping all your protein away. Consider storing the membrane in the fridge in just PBS to see if this slows the leeching process. I have done this for almost a week and I was still able to probe using good antibodies.

You could also be losing it all by stripping too harshly. For stripping I really enjoy this product: https://www.thermofisher.com/order/catalog/product/21059#/21059

It is more gentle than a stripping buffer that contains BME and/or SDS and it can be re-used for multiple strips.

Lastly, make sure you are loading 20-30 ug total protein. If you are loading very little protein, it is easier for what little signal you have to fade more quickly.

Have you tried to directly reprobing your membranes with new primary antibody overnight, without storing it in PBS-T inbetween?

You could try to stain one of your "dead" membranes with Ponceau S again to make sure your protein hasn't leeched out during storage.

Make sure your membranes do not dry out during storage, e.g. they are completely covered in your storage buffers. Otherwise you have to reactivate them again. They may also dry up during/after development.

If you absolutely must store PVDF membranes, in my experience just letting them dry on a clean paper towel and storing them in a folder can work fine.

Thanks for your reply Max Schauer !

Yes, I often incubate with new primary antibody directly without storing in PBS-T inbetween. Sometimes this works, but mostly I have the same issues and get no signals at all.

I have also tried to stain my membranes after storage to see if I have lost proteins, and the ponceau stain always looks OK.

I usually never let my membranes dry out.

Also thanks for your answer Evan Kerek :)

I actually never strip my membranes as I am affraid it will affect the proteins.

I will try loading more protein (20-30 µg) as I actually only load 10 µg right now! And I will try storing in PBS without Tween.

However, do you have any idea, why I can loose signal already on second probing, when I probe directly without storing in PBS-T?

Sophie Emilie Blomgren Ambjørner If you are reprobing without stripping are you cutting the membrane to get rid of the signal from the 1st target then reprobing for the 2nd target on the remaining piece? Since western blots are semi quantitative, the signal you see is in reference to the darkest thing on the blot. If you probe Target #1 and it is a stronger antibody than Target #2 and you don't strip off Target #1 first, you may not see much signal from Target #2 because it is lower relative to Target #1. A couple of ways to navigate this is to do fluorescent western blotting where Target #1 is uses a rabbit secondary on a red alexa fluor while Target #2 uses a mouse secondary on a blue or green alexa fluor. This enables both targets to be probed simultaneously but fluorescent western blotting has its own limitations you can read up on.