Lost my proteins ! - (Jun/15/2009 )

I ran a mini-gel for the first time today and when I took the cassette out of the transfer, there were no marker bands on either the gel or the membrane. I used PVDF membranes and 8% gel. I can see the protein ladder faintly on one of the whatman papers from the cassette but lost track of which side it came from. I ran the transfer( in 4ºC room) initially at 400 mA; decreased to 300mA after 30 min as it was getting too hot, then ran it at 300 mA for 2 hrs ( I had initially been recommended a 300 mA 3 hr transfer, but was in a little hurry). I am pretty sure I aligned the electrodes correctly, I rechecked it 2 or 3 times before starting. But now I am confused.

Did I switch the elctrodes or did the proteins run out of the gel, or what?

1. Did you set up the transfer correctly- as in the PDVF membrane and gel were in right orientation for transfer?

2. The transferring mA seems high, and with the time you transferred there is a possibility that the proteins when through the membrane and into the sponges in that time.

I usually run blots as 0.8mA/cm2 for 1 hr or slightly longer for higher molecular weight proteins (thats semi-dry blotting)

And wet blotting is 30V constant for 1hr (thats using invitrogen NuPage system though)

higher molecular weight proteins- I transfer for longer (2 hours), or overnight at 4C, but drop the V or mA (depending on the system) for long transfers

I guess that you were blotting too long.
I use the wet blot system from biorad and make the transfer for 1 hour at 100V.

I think i switched something the other way in the cassette; I ran the gel today again and did 300 mA 3 hr 4ºC transfer; have nice bands on the membrane. Fingers crossed !

We use Biorad mini trans-blot system, and used 300 mA because the lab next door does that. Looks OK. Will see.