I was wondering if anyone else has used the iBlot PVDF membrane stacks and noticed uneven and/or relatively high autofluorescence / background when used for fluorescence Western blotting? I am using the Odyssey system, and have heard that despite the fact that different vendors claim that their PVDF membranes show low autofluorescence/background, that they can show relatively high lot to lot variability and background? I've heard that the Millipore Immobilon FL membranes are the best for this purpose? Any experiences?
Thanks much,
Nat
When I used the Odyssey and Immobilon-FL membranes, I found that doing the old-school, full-wet transfer worked best to decrease autofluorescence. Additionally, don't use milk-based buffers, because those also add a significant amount of autofluorescence. The semi-dry iBlot transfer didn't work nearly as well for all of my proteins as the full-wet transfer did. I hope this helps.
Thanks John! I haven't thought about a wet transfer yet, I have been using the iBlot system, good to know!
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence