9 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.

[maizeprince]

I also have this problem, and I am going to soak the gel (after electrophoresis) in 2% SDS for 10min before transferring and the following immunoblotting. Is there any better suggestion available? thanks

[mdfenko]

maizeprince, on 10 December 2012 - 08:20 PM, said:

I also have this problem, and I am going to soak the gel (after electrophoresis) in 2% SDS for 10min before transferring and the following immunoblotting. Is there any better suggestion available? thanks

you're going to soak in 2% sds prior to transfer? you can just add up to 0.05% sds in the transfer buffer (with 20% methanol). that would be sufficient for transferring large proteins.

[maizeprince]

Let me clear my work more. I am going to do immunoblotting after native gel electrophoresis and transferring. To avoid the poor recognition of antibody due to the possibility of epitope embed in the complex, I want to use SDS to break and loose the complex, and let the epitope available for antibody binding. This is just a quick and dirty job. Of course, 2D will be the next choice if above test is not workable. So, 0.05% is enough? thanks for the kindly reply.

[mdfenko]

0.05% should work. any more and you are taking the chance of interfering with binding to the membrane (don't forget the methanol to strip the sds from the protein during transfer).

soaking the gel with transfer buffer should be sufficient to allow the sds to denature the protein.

[cancerbio22]

Hi, I have this same problem and have tried the suggestions on this page.  With 0.05% SDS in the transfer buffer my complex was still not visible (A positive control antibody to a different complex worked on the other hand).  With 2% SDS preincubation of the gel prior to transfer, I also did not see my complex.  Has anyone tackled this before?

[mdfenko]

cancerbio22, on 21 April 2013 - 10:11 AM, said:

Hi, I have this same problem and have tried the suggestions on this page.  With 0.05% SDS in the transfer buffer my complex was still not visible (A positive control antibody to a different complex worked on the other hand).  With 2% SDS preincubation of the gel prior to transfer, I also did not see my complex.  Has anyone tackled this before?

have you confirmed that your antibody is good with denatured protein of interest?