help me ! dying about my western blot - (Jul/25/2006 )

hi kathy,
can u look at my attachment n say whether the marked region is membrane alone or it is covered with gel during transfer?

if it is membrane alone, i wud expect some problem after transfer.

regards
payeli.

Commercial gels and reagents go off all the time.

I use pre-cast gels and buy all my buffers. We've had problems with gels not runnig properly (though nothing like yours). It look s as though you can see smearing of the samples down the edges of each lane. So this is either contamination or something is past it sell-by date.

What is your sample? Could it have gone off? Aggregation, etc?

the sample is fine, because I let my collegue to run my sample and it shows fine.
I really could not think of anything, rule out everything.
the precase gel you said could be, today a collegue use it, we will know.

maybe the antibody solution is not mixed very well,

and the other reason is that maybe when you block the membrane or incubate with antibody, you did not shake well.

i think you can use more solutions and make sure all the solution are mixed well, use fresh running and transfer buffer next time, when you wash the membrane, use large volume of buffers.