Putting more than one membrane in a container... - Western (Feb/09/2005 )

Hi everybody,

My question is:

After SDS-PAGE I usually transfer onto PVDF membrane. Let's say I ran two gels and then transfered those gels onto 2 PVDF membranes. Can I put the two PVDF membranes in the same container and do blocking, first and second antibodies, plus washing in that container? (I am probing both membranes with the same antibodies.)

I ask this because when I do this, it seems that the membranes stick together, so I am worried that when I put the first antibody in the container (about 5ml of diluted antibody), the antibody will not reach the bottom membrane since the membrane on top is stuck to the bottom membrane. However, a colleague told me that this is no problem actually, because the antibody will seep in even though the membranes look like they're stuck together.

I hope I'm being clear here. Anyone think this is no problem?

We do block and incubate with the antibodies 2 membranes at the time... there's usually a film of liquid between the membranes which is enough, for us at least, to see equally good signal from both membranes.

Simon

I make a v-shaped creased ridge in a piece of parafilm and lay it down in the container before adding my blots or liquid. Works well enough because the parafilm doesnt move but the liquid can move freely between the two sections and the blots never touch each other.