Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting

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Abstract

The discovery of Green Fluorescent Protein (GFP) and its derivatives has revolutionized cell biology. These fluorescent proteins (FPs) have enabled the real-time observation of protein localization and dynamics within live cells. Applications of FP vary from monitoring gene/protein expression patterns, visualizing protein-protein interactions, measuring protein stability, assessing protein mobility and creating biosensors. The utility of FPs also extends to biochemical approaches through immunoblotting and proteomic analyses, aided by anti-FP antibodies and nanobodies. FPs are notoriously robust proteins with a tightly folded domain that confers a strong stability and a relative resistance to degradation and denaturation. In this study, we report that various green, orange and red FPs can be maintained in a native, fluorescent state during the entire process of protein sample extraction, incubation with sample buffer, loading and migration on SDS-PAGE with only minor adaptations of traditional protocols. This protocol results in the ability to detect and quantify in-gel fluorescence (IGF) of endogenously-expressed proteins tagged with FPs directly after migration, using standard fluorescence-imaging devices. This approach eliminates the need for antibodies and chemiluminescent reagents, as well as the time-consuming steps inherent in immunoblotting such as transfer onto a membrane and antibody incubations. Overall, IGF detection provides clearer data with less background interference, a sensitivity comparable or better to antibody-based detection, a better quantification and a broader dynamic range. After fluorescence imaging, gels can still be used for other applications such as total protein staining or immunoblotting if needed. It also expands possibilities by allowing the detection of FPs for which antibodies are not available. Our study explores the feasibility, limitations, and applications of IGF for detecting endogenously expressed proteins in cell extracts, providing insights into sample preparation, imaging conditions, and sensitivity optimizations, and potential applications such as co-immunoprecipitation experiments.

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  1. Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reviewer #1

    Evidence, reproducibility and clarity

    Sanial et al. carefully analyze the use of in-gel fluorescence as an alternative to immunoblotting. The authors show that simple modifications of common protein extraction protocols can preserve (to varying extents) fluorescent proteins in their native, fluorescent states. This can be exploited in different applications for in-gel fluorescence quantification, bypassing immunoblotting. The experimental results are clear, showcasing the ease and linearity of in-gel fluorescence quantification.

    In my opinion, the trick of this approach is also potentially its main drawback, the partial denaturation …

  2. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    In this paper, Sanial et al present in-gel fluorescence detection (IGF), a method that allows the direct detection of fluorescent proteins from SDS-PAGE gels with minimal adaptation of existing protocols. The authors test a range of fluorescent proteins routinely used, especially when working with yeast, and describe their behavior in IGF. They identify heat-induced denaturation of fluorescent proteins as the main component influencing their assay and systematically test this on a selection of fluorescent proteins. Next, they compare the detection limit and the linearity of the signal between IGF and chemiluminescence, showing that …

  3. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The present manuscript "Direct observation of fluorescent proteins in gels: a rapid cost-efficient, and quantitative alternative to immunoblotting" describes a method how to visualize bands of fluorescent protein fusions onto a common SDS-PAGE without antibody staining. It is based on ability of GFP-like fluorescent proteins (FPs) to retain their fluorescence under conditions of SDS-PAGE if step of extensive heating (boiling) of protein sample is omitted. This property of FPs is not novel; it was known for more than 20 years (for example, see Fig. 2 in Yanushevich et al. FEBS Lett. 2002 Jan 30, 511:11-4; Supporting Fig. 7 in …

  4. Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons


    Referee #1

    Evidence, reproducibility and clarity

    Sanial et al. carefully analyze the use of in-gel fluorescence as an alternative to immunoblotting. The authors show that simple modifications of common protein extraction protocols can preserve (to varying extents) fluorescent proteins in their native, fluorescent states. This can be exploited in different applications for in-gel fluorescence quantification, bypassing immunoblotting. The experimental results are clear, showcasing the ease and linearity of in-gel fluorescence quantification.

    In my opinion, the trick of this approach is also potentially its main drawback, the partial denaturation conditions. I think the manuscript …