If I don't have time for immediate processing and signal detection, I keep the membrane in TBS-T at 4 C for less than one week.
My friend said this was over cautious. He usually keeps them over 3 weeks and is still able to get good bands (for even proteins of low abundance).
I suggest that if don't have time for immediate processing the membrane, leave it in secondary antibody rather than TBS-T. If it remains in secondary antibody for months, no problem at all, as after secondary antibody has overlapped the primary antibody, no further sites remain for secondary antibody attachment, and at this point it just keeps the membrane wet. So whenever you find time, remove secondary antibody, and wash the membrane with TBS-T for 40-60 minutes, and then get the signal by ECL or the suitable protocol you use.
Best Regards!
I would personally not leave the membrane in TBS-T due to the tween. I would suggest you keep the membrane in between transparent folios and tape them shut as not to dry out the membrane. They can last in the fridge under that conditions for up to 3 weeks before applying primary antibody.
Hi Lei, Good to see your question. In our lab, we normally put the wet membrane in a plastic sleeve and seal the edge of the sleeve. Then freeze the membrane at -20C, which can be re-probed whenever you need to probe the membrane. You just need put the membrane in the stripping buffer as normal, then block and probe for primary and secondary. Good luck.
It depend of the type of membrane.
For PVDF, after transfert, you could dry it 20min at RT on a wattman paper. After, I put the membrane into 2 plastic film and seal it. You could keep your membrane 2-3 weeks at -20C. For nitrocellulose, do not dry the membrane.
To use your PVDF membrane, you unfreeze your membrane, put it few second in ethanol and 10min in water. You perform the blocking steps, first and second antibodies and revelate your data.
After each freezing, you need to block and due the both antibodies.
As most of people already said, I wouldn´t leave it in TBS-T, I would do it in TBS in order to avoid the Tween effect.
In respect to the storage, in our lab we use PVDF membranes, if Im going to detect them in one week or less we store it in TBS. Fore longer time, we dry it and store it between two plastic films, as some fellow have already explained.
Thank you very much for everyone's opinion. I am using regular nitrocellulose membrane, and I try to keep them in TBS-T for only than a few days.
For long term storage, I do air dry, seal in sheet protector and freeze at -80 C. I am not sure whether the air dry step helps to stabilize antigens on membrane, or puts the membrane in danger. Since, most protocol suggests that during western blot, we should avoid the membrane to be dry.
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