Blotting time and voltage for a 20kd protein - (Jan/18/2006 )
What is the minimum time, voltage and current required to transfer proteins of less than 20 kDa. I am facing problem in that. I just want to have your opinions.
I use 150 mA one hour for a 8x6 cm gel.
it's absolutely fine up to 50 kDa at least.
I use 1 mA/cm x cm for one hour
Works fine for my protein (23 kDa)
Depending on if you are using wire of plate electrodes.
Based on what you write, 1 hr, 100 volts (I blot on volts not amps like the others) is fine.
If you are paranoid about losing your protein, use two sheets of nitrocellulose, if it blows thru the first, the second will catch it. Ponceau S stain both to verify which sheet got it.
I transfer a 6.5kDa and a 14kDa protein for 1hour at 100V. Both Coomassie and western blotting works fine for me. Ive dropped this time to 45mins to work well too.