Hi, just wondering if drying a PVDF membrane after transfer will effect my antibody binding, if I forgot to re-wet it with methanol before blocking?
There might be issues. You'll notice PVDF doesn't absorb solutions in a consistent pattern unless pre-wet with methanol. If your blot ends up messier than normal, that'll likely be why. But you can always restart the process, with some signal loss.
Hi,
After the membrane has been wetted with buffer, do not allow it to dry (white spots will form where the membrane is dry). Protein will not bind to the dried membrane, and dry spots will not rewet in aqueous solutions. Due to the hydrophobic nature of PVDF,it's required to pre-wet in methanol in order to equilibrate membrane in the transfer buffer and also help in the protein binding capacity.It's worthy to note that if PVDF membrane is not pre-wetted, protein won't bind to the membrane.
All the best!
Tola
Actually:
Dried membranes are non-active (because are hydrophobic) and will bind Abs ONLY in the sites occupied by proteins. It is the principle of Rapid immune-detection (no blocking, Abs are diluted in non-blocking buffer, ½ time incubation: 1ary just 1h, 2ary just ½ h). It is recommended ONLY for high abundance targets.
- Badges
- Science method