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.
Hello!
I have also done quite some Western Blots in my life and I never faced the problem you are describing. However I had similar issues when doing IHC: with higher room temperatures in summer, I got far much background that was not distinguishable from the real signal. So, yes, room temp seems to have an impact on antibodies - but in my case it was higher activity.
What if you incubate your antibodies over night at 4°C instead of 1-2 hours RT? And keep your 10x buffers in the fridge, then prepare fresh dilutions on the day you need them? Are you using wet or semi-dry transfer? I found that wet transfer works better if I put the whole tank in a box filled with ice!
Good luck!
Thank you Sarah! I usually incubate antibodies at 4ºC over night... Regarding buffers I will do what you recommended ;)
Yes, it makes more sense to increase background with higher temperatures, but in my case the signal is lost...:( This is why I did the question, maybe it will affect antibodies or buffers or membrane...
Thank you for your help!
Dear Elena,
We face this problem every summer, with 28-30°C in the lab, WB becomes frustrating. If you have air conditioning in your lab room, turn it on (especially during RT incubation and wash), keep stock solutions of TBS and PBS in fridge (check for precipitates) and prepare fresh working solutions every day. Usually primary antibodies are not a big problem since are employed at 4°C , but they will probably last less time. To increase their stability prepare them in 3-5% BSA and 1x NaN3. Secondary antibodies are quite heat-sensitive, and the HRP will likely generate lot of background if you incubate over 25-26°C. In addition, keep clean and refrigerated the ECL and the developing and fixing solutions as well (if you use them).
Good luck
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