Hello, everyone,

I've been doing Western-blocks for years.

For a few days now I have had problems, even with "easy" antibodies such as GAPDH. According to my findings, proteins are in good condition (Ponceau shows defined bands) and the transfer is efficient, but I don't detect any signal.

Looks like something stopped working.

On Wednesday I got signal by changing all the reagents, but the next day I repeated it and got no signal.

This problem has coincided with an increase in room temperature (summer is coming). It seems I can't recycle first antibodies, when I usually recycle GAPDH several times. And the reagents also seem to have to be prepared fresh the same day.

Has anything similar happened to anyone?

Thank you very much.

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