Testing the primary antibody - Is the secondary antibody recognizing the primary (Jun/25/2008 )
Hi
We are making western blots to detect a cellular protein.
Unfortunately we do not detect anything.
For troubleshooting we are planning to test, if the secondary antibody binds the primary.
My question, is dot blot analysis suitable therefore? Is it possible to dot the primary AB on a PVDF Membran. And what should be the conditions that the primary AB binds to the Membrane without denaturation (so that it is accessible for the secondary?)
Thank you
-AntonBruckner-
QUOTE (AntonBruckner @ Jun 25 2008, 12:23 AM)
Hi
We are making western blots to detect a cellular protein.
Unfortunately we do not detect anything.
For troubleshooting we are planning to test, if the secondary antibody binds the primary.
My question, is dot blot analysis suitable therefore? Is it possible to dot the primary AB on a PVDF Membran. And what should be the conditions that the primary AB binds to the Membrane without denaturation (so that it is accessible for the secondary?)
Thank you
We are making western blots to detect a cellular protein.
Unfortunately we do not detect anything.
For troubleshooting we are planning to test, if the secondary antibody binds the primary.
My question, is dot blot analysis suitable therefore? Is it possible to dot the primary AB on a PVDF Membran. And what should be the conditions that the primary AB binds to the Membrane without denaturation (so that it is accessible for the secondary?)
Thank you
what do you mean by denaturation? activation of the membrane?
water-based fluids hardly soak in PDVF, NC may be used, or a dot-blot apparatus; to prevent direct binding of sec Ab you have to block as for Western blot
-The Bearer-