Fixation to PVDF membranes - (May/26/2008 )
Has anyone ever tried fixing protein to a PVDF membrane. I am essentially doing a far western blot trying to identify what my AP fusion protien binds to, however i need to do a 65 deg heat treat to remove endogenous AP and this appears to effect my Receptor-ligand interaction. Im thinking of fixing before heat treat with paraformaldehyde/acetone fixative such as what you would use in ISLB. Think this would work? ever tried it anyone? any advise or insight would be helpful.
J
-jturne5-
QUOTE (jturne5 @ May 26 2008, 01:38 PM)
Has anyone ever tried fixing protein to a PVDF membrane. I am essentially doing a far western blot trying to identify what my AP fusion protien binds to, however i need to do a 65 deg heat treat to remove endogenous AP and this appears to effect my Receptor-ligand interaction. Im thinking of fixing before heat treat with paraformaldehyde/acetone fixative such as what you would use in ISLB. Think this would work? ever tried it anyone? any advise or insight would be helpful.
J
J
is your AP-fusion protein separated by SDS-PAGE and then blotted, or would you like to add your AP-fusion protein as solution to a Western blot?
-The Bearer-
QUOTE (The Bearer @ May 27 2008, 04:54 AM)
QUOTE (jturne5 @ May 26 2008, 01:38 PM)
Has anyone ever tried fixing protein to a PVDF membrane. I am essentially doing a far western blot trying to identify what my AP fusion protien binds to, however i need to do a 65 deg heat treat to remove endogenous AP and this appears to effect my Receptor-ligand interaction. Im thinking of fixing before heat treat with paraformaldehyde/acetone fixative such as what you would use in ISLB. Think this would work? ever tried it anyone? any advise or insight would be helpful.
J
J
is your AP-fusion protein separated by SDS-PAGE and then blotted, or would you like to added your AP-fusion protein as solution to a Western blot?
The PVDF membrane is incubated in my AP fusion protein after either native page separation or dot blotting.
-jturne5-