I recently got new secondary antibodies but didn't realize they needed to reconstituted prior to use (first time getting fresh ones myself) until after I had finished washing my membrane with TBS-t. How long can my membrane sit in TBS-t? There is 30 minutes between end of last wash and when the secondary antibodies will be ready to use. Should I place the membrane in TBS while I wait?
Taylor McGillis Based on the search results, a PVDF membrane can be stored in TBS-T buffer for a short period of time while waiting to use reconstituted secondary antibodies:
- PVDF membranes can be stored wet at 4°C in TBS-T buffer containing sodium azide to prevent bacterial growth, but the sodium azide should be thoroughly washed out before use as it inhibits HRP activity.
- It's important to rehydrate the PVDF membrane with methanol prior to use (blocking and incubating with antibody).
- Storing the membrane in TBS-T for 30 minutes while waiting for the secondary antibodies to be ready should be fine. The key is to not let the membrane dry out.
To summarize:
- Place the PVDF membrane in TBS-T buffer while waiting for the reconstituted secondary antibodies.
- Make sure the membrane remains wet and doesn't dry out.
- When ready to use the antibodies, rehydrate the membrane briefly with methanol first.
- Proceed with blocking and incubating the membrane with the antibodies as usual.
The 30 minute wait time in TBS-T should not negatively impact the Western blot. Just avoid letting the membrane dry out and rehydrate it with methanol before proceeding with the antibody incubation step.
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