Sparky sparkster at writeme.com
Wed Feb 23 10:01:18 EST 2000

Hi Panagiotis,

> I am running a slab ief gel, using ph range 4-6,5. I am trying to
> transfer my ief seperated proteins to NC membrane but there is no
> result.
> Before trasnfer i equilibrate my ief gel in : 192mM Glycine, 50mM
> Tris, 0,1%SDS, 20%Merhanol and i am using the same buffer for
> trasfering.
> Next im staining me Nc membrane using Ponceau, but there is no protein
> on membrane. Finally, i have no signal, using my monoclonal antibody
> and hyperoxidase.

This should be working. What are the electrical parameters of your
transfer protocol? We've had success blotting to nitrocellulose in a
semi-dry transfer cell at 10-15 V, >300mA for 70 minutes. The transfer
buffer you use seems fine: we use the same thing. What exactly are you
seeing with the Ponceau stain? Does nothing on the membrane stain, or
does the entire membrane turn red?

"Violence is the last refuge of the incompetent."--Isaac Asimov
"A foolish consistency is the hobgoblin of little minds."--Ralph Waldo

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