Apologies if this has been asked a few times before but it's a recurrent problem with me and seems to rear it's ugly head a few times a year.
Blank membranes - I mean completely blank - no MWM or anything!
I normally load between 50-100ug of protein, run at 40mA until loading dye runs off gel, transfer overnight, ponceau (which incidentally always shows bands), wash off with dH20 quickly then into PBS-T, block for 1 hour with 5%milk powder in PBS-T, primary also in 5%milk (either overnight or 1 hour at RT), wash x 3 with PBS-T, then into secondary for 1 hour before washing x 3 with PBS-T and developing with ECL.
There seems to be no rhyme nor reason for this - ie one day, I get bands and another day nothing!
Anyone shed any light?