I'm trying to transfer a low molecular weight protein from a 15% SDS-PAGE gel to a 0.45um PVDF membrane. I've heard many people suggest using a 0.20 um membrane, but many others suggest that it is possible on a 0.45um membrane. I typically transfer at 100V and 400mA for 1 hour or 114 mA at 35V overnight (~16h). Does anyone know which would promote better transfer for low MW proteins (~7kD). I have ponceau S staining results for the 1 hour transfer and they look good, but I don't see any low molecular weight bands for my samples (C6/36 cells) but I always have a good color band for my ladder at 10kD.
hi dave,
indeed, 0.22um membranes are better. i work with a 10kd protein and my blots only started to work with 0.22um membranes. you can try to reduce the transfer time for 30min. moreover, you can try more sensitive revelation methods such as odissey or supersignal west dura from pierce.
good luck!
Hi! I completely agree with Fabianno. You can also check if you don't have any color bands from your marker on the Whatman's paper (the small one of course). It's an easy way to see if the transfer time isn't too long.
Good luck! :)
Thank you both. Yeah, the 10kD band in my marker is always faint and sometimes not visible at all, even when I use two membtanes sandwiched togerther suggesting that it's not "blowing through". I've heard that the SDS in the PAGE gel (no SDS is used in the transfer buffer) will bind stronger to smaller proteins thus reducing transfer rate to the membrane. So could it be possible that a longer transfer is necessary? Ive been suggesting the use of the .20 micron PVDF membrane to the powers that be for a while. Although, as mentioned previously other researchers indicate that it's possibe. Thank you both for your advice.
Do you still see the 10kDa marker band in the gel though? I'd think even if you aren't getting it on a second membrane it could still be blowing through. I do a 350mA transfer for 40 min for my 25kDa protein, and that seems to work well.
Alternatively you could use a different membrane depending on the properties of your protein? Nitrocellulose or nylon?
- Badges
- Science topic
- Similar topics
- Gels