SDS antigen retrieval and GFP signals

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SDS antigen retrieval and GFP signals

Postby dreamwest » May 14 2007 1:50 pm

Hi,

I am wondering if anyone has experience using SDS (1% SDS for 5min) to retrieve antigens. I can get very good staining using this method, but I am not quite sure if it's artifact or not.

Also, does anyone know if SDS antigen retrieval would affect GFP signals or not. I am transfecting my cells with some GFP construct, but in order to stain for another antigen, I usually do SDS retrieval to get the best staining result.

Thanks in advance.
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Re: SDS antigen retrieval and GFP signals

Postby lupulin » Jul 13 2010 10:03 pm

In my experience doing immunofluorescence on GFP-expressing cells I was able to see nice GFP fluorescence in cells that were briefly fixed with formalin. However, if I used heat-induced epitope retrieval on these cells in order to pre-treat for an additional antibody stain I lost the GFP signals. I've also not been able to retrieve GFP signals in tissues that had been routinely fixed and processed into paraffin blocks. It appears that treatments involving heat and/or organic solvents are likely irreversibly denaturing the GFP protein, thus killing it's fluorescence. SDS is a strong protein denaturant, so my guess is that it will kill your GFP signals. Are you sure you need to use SDS in pre-treatment for this additional antibody stain? I would try other, less harsh, pre-treatment conditions. Many antigens become stainable using brief treatment with solutions of gentler non-ionic detergents such as Triton or NP-40. These may preserve the GFP and alllow detection of the other target. Alternatively, there are good anti-GFP antibodies available, so you could work out dual IF staining.
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