Hi all,
I am looking into switching from nitrocellulose membranes to pvdf membranes for LC3 detection in western blotting. Can I use ethanol (as opposed to methanol) to hydrate my pvdf membranes before transfer? In addition, can I use ethanol in my transfer buffer when doing a western blot with pvdf membranes, or do I need to stick with methanol?
Thank you for your help! I really appreciate it!
Yes, it is applicable.
Leave your membrane for 30 sec in an absolute ethanol, then immerse it in a 25% ethanol transfer buffer.
PI: from my experience ethanol does better than methanol especially for HMW proteins.
Hello Melissa Rogers
As rightly mentioned by Mohamed Khashan , you may use ethanol or even isopropanol to activate the PVDF membrane. It is not always methanol as most of the researchers usually do. If you are using ethanol to activate the membrane, then you need to add a few extra minutes. You may wet the membrane in ethanol for a few minutes (2-3 mins), and then equilibrate the membrane in transfer buffer for a few minutes (until it sinks).
Ethanol can be used instead of methanol in transfer buffer with minimal impact on transfer efficiency. You may have to use 10% ethanol in transfer buffer. Ethanol is more hydrophobic than methanol as ethanol possesses an additional hydrophobic carboxyl group. So, you will also need to equilibrate with the transfer buffer for a longer time.
Best.
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies