western blot transfer - what's your opinion? (Feb/23/2007 )
I have a new protein marker that can be showed in x-ray film by using ECL kit. But this marker does not transfer in 100 V for 1 h or 30 V overnight (while the proteins transfer); It is written in its protocol to transfer in 200 V for 2 hours, but I have'nt read this in any other protocol. What's your opinion? Shall I transfer my proteins in this condition? Can proteins pass the blot ?
Thanks in advance.
Thanks in advance.
2 h are pretty long for semi dry blotting if you use semidry and may enter total dry at the end; proteins can pass the filter especially for nitrocellulose, more difficult for PVDF; I would think of if your standard proteins are the ideal solution for reference
How are you transferring? I use the wet/tank transfer system and 2 h at 100v would be overkill in my experiments. Depending on the size of your protein of interest you may risk it passing through the membrane under those conditions ( my protein of interest is 20kda and I found that it was passing through 2 pieces of PVDF membrane when I transferred under standard conditions of 100V for 1h!)
So what is reccommended voltage for transferring low MW proteins. I usually transfer at 100V for 90 min for all proteins.
In our lab, transfert are set with mA instead of volts. So we transfert at 40 mA per gel, for 1 h.
for a 50 kda protein, I transfer 400 mA (2 gels) for 1 hour 30 min (although I suspect that less time is enough) in a wet/tank system, and the same protein in a semidry system (much better, faster and saves time and buffer) I transfer for 30 min at 15 V (2 gels).
I found (after lots of optimising) that 1hr at 40V (wet transfer) works well for my 20kda protein. I generally do westerns on tisuue homogenates and could pick up ubiquitin (~8kda) using standard conditions(1hr at 100V) - so I guess it depends on the abundance of your small protein in your sample as to whether you can pick it up under standard contitions.
as an aside some one told me that transferring at constant voltage was better for quantification than transferring at constant amps - has anyone got an opinion on this?