ief-electroblotting

[Sparky]

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?

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


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