3 replies to this topic

[Bobby007]

I am doing a Blue Native gel followed by a Western to detect protein complexes.

My detection antibody only recognizes a denatured epitope so I have to denature proteins after the gel is done. I tried doing that "in gel" by soaking in 2% SDS and DTT before transferring and the Western was blank. I can clearly see proteins on the membrane by Ponceau S, just no signal. Any recommendations to denaturing proteins on a PVDF? I have read that detergents can wash proteins off of PVDF easily so I can't do anything too harsh... Help!

[D.Huckab]

Bobby007, on Aug 7 2009, 05:12 PM, said:

I am doing a Blue Native gel followed by a Western to detect protein complexes.

My detection antibody only recognizes a denatured epitope so I have to denature proteins after the gel is done. I tried doing that "in gel" by soaking in 2% SDS and DTT before transferring and the Western was blank. I can clearly see proteins on the membrane by Ponceau S, just no signal. Any recommendations to denaturing proteins on a PVDF? I have read that detergents can wash proteins off of PVDF easily so I can't do anything too harsh... Help!

Is performing a 2D electrophoresis out of the question? You could cut out the gel strip the band is in, denature the strip by boiling in SDS, Mercapto, etc... overlay it on an SDS-Gel, run it, transfer and then blot.

[Bobby007]

D.Huckab, on Aug 9 2009, 07:14 PM, said:

Bobby007, on Aug 7 2009, 05:12 PM, said:

I am doing a Blue Native gel followed by a Western to detect protein complexes.

My detection antibody only recognizes a denatured epitope so I have to denature proteins after the gel is done. I tried doing that "in gel" by soaking in 2% SDS and DTT before transferring and the Western was blank. I can clearly see proteins on the membrane by Ponceau S, just no signal. Any recommendations to denaturing proteins on a PVDF? I have read that detergents can wash proteins off of PVDF easily so I can't do anything too harsh... Help!

Is performing a 2D electrophoresis out of the question? You could cut out the gel strip the band is in, denature the strip by boiling in SDS, Mercapto, etc... overlay it on an SDS-Gel, run it, transfer and then blot.

Thank you.
I was trying to avoid doing a 2D and was hoping that I could salvage this experiment by treating the membrane somehow. Any way to denature post-transfer and not wash off my material?

[mdfenko]

you should be able to denature the protein on the membrane without losing too much, just soak in a solution similar to one you would use to soak for a 2d gel.

sds is used to strip antibodies from the blot, not the bound protein.