I used to activate my PVDF with methanol for 5 minutes, then I did transfer as usual. But these days twice I got the same results, which is there is a block white marks in between after I finished my fast-green staining. I do believe those are not bubbles formation.
Some student suggest me to wash the PVDF first with water then continue transfer and so on.
I confuse, what's wrong with my technical transfer. I have never facing this situation before.
Hello Wenny Ambarsari
You need to activate the PVDF membrane by wetting the membrane in 100% methanol for 30-40 seconds and soak it briefly in distilled water and then equilibrate the membrane in transfer buffer for a few minutes (until it sinks).
Please note that you need to handle the membrane carefully, ideally with rounded tweezers to avoid scratching or puncturing the surface. Avoid touching the membrane with your fingers. Proteins on fingers will block efficient transfer and create dirty blots.
Best.
Hi Wenny
As for me, I activate the PVDF for 2-3 mins in methanol and then soak briefly in WB transfer buffer for 30sec - 1min.
Hi Wenny
I used to activate PVDF FOR 20-30 seconds in methanol and one minute in transfer buffer. This looks like, you have exposed the PVDF membrane to air after transfer. Kindly avoid that and try.
All the best@ Wenny
With regards
S.Dharani
Hi Wenny,
I usually soak the membrane in methanol for 30 seconds, water for 2 minutes and then cold transfer buffer for no longer than 10 minutes. Try to avoid touching the membrane - always use tweezers and gloves. However, from your image, I think air bubbles are causing your problem. When you are constructing your transfer cassette try using a roller on the sponges/blotting paper with your membrane in and blot in the middle to get rid of these.
Good luck!
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