Ponceau staining - Appearence of bands (Mar/13/2010 )
I have been using Western blotting (semi-dry) for a month.
Membrane - PVDF
Transfer Volts - 25
Current – 250 A
Time – 80 min
Recently, while performing one of my experiments, I stained the membrane using Ponceau Red. However, I couldn’t observe the bands/staining of the proteins on the side of the membrane, where contacted with the gel, but on the opposite side. I would be glad if anyone could explain the reason why I saw like that. Also, please tell me how I can rectify this problem in future. Thank you!
what about after developing the membrane? did you see your band of interest at all?
I prefer using pre-stained markers to avoid ponceau staining. If I see the marker on the membrane then I'm sure my proteins transfered to the membrane. Ponceau staining wastes your time.
For Nitrocellulose membranes it's recommended to use the shiny part to face the gel. Unlike Nitrocellulose, you can use both sides of PVDF. so I'm not really sure why this happened.
last time I had some problem with Thermo membranes. They became transparent after semi-dry electroblotting. Then I changed to Millipore and it works fine.
What was your sample by the way?