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

  1. Matthieu Sanial
  2. Ryan Miled
  3. Marine Alves
  4. Sandra Claret
  5. Nicolas Joly
  6. Véronique Proux-Gillardeaux
  7. Anne Plessis
  8. Sébastien Léon

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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. Version published to 10.1101/2024.05.31.594679v2 on bioRxiv
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    Reply to the reviewers

    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 conditions. I think the manuscript could be strengthened with more extensive benchmarking of the approach and further discussion of potential caveats as detailed below.

    Major points:

    1. Protein abundance in the original GFP library (and in other FP-tagged libraries constructed in the meanwhile) have been quantified using fluorescence (flow cytometry, microscopy, colony fluorescence) (Ho et al. 2018 10.1016/j.cels.2017.12.004, Weill et al. 2018 10.1038/s41592-018-0044-9, Meurer et al. 2018 10.1038/s41592-018-0045-8). This provides an opportunity to significantly strengthen the manuscript (where most of the test have been done using two abundant cytosolic proteins Bmh1 and Hxk1) if the authors could apply their approach to a representative fraction of the yeast proteome (sampling from such libraries FP-tagged proteins that differ in abundance, localization, membrane vs cytosolic/nuclear, subunits of large stable complexes vs proteins not part of complexes, etc.) and compare their quantification with previous relative abundance estimates. This information would also help future users in case protein-specific issues are identified.

    Indeed, Hxk1 and Bmh1 are quite strongly expressed (41,000 and 65,000 copies/cell, according to SGD, ____www.yeastgenome.org____). In the course of our experiments we were able to detect proteins with a much lower expression level (eg. Reg1, 4000 copies/cell). We have selected a number of proteins based on their expression level as detailed in SGD, ranging from 700 to 75000, and plan to detect the signal by IGF and compare it with published data on absolute protein quantifications for ecah protein. However, this will take a bit of time as each gene must be tagged with EGFP – we cannot use the GFP-S65T from the GFP collection which is poorly amenable to IGF because of its sensitivity to denaturation, as we show in our manuscript.

    The authors discuss several drawbacks, including the change in apparent molecular weight compared to denatured proteins; differential recognition of folded vs denatured proteins by antibodies.

    Other potentials drawbacks should be discussed. For instance, the need of additional steps post-fluorescence imaging for signal normalization against a loading control; the need of antibodies and immunoblotting to decide on the best denaturing temperature for a specific protein or FP tag; complexity of the native protein extraction protocol compared for example to alkaline lysis followed by TCA precipitation (Knop et al. 1999 PMID: 10407276).

    • Regarding the the need of additional steps post-fluorescence imaging for signal normalization against a loading control – this doesn’t take extra time for people using gels with protein stain included in the gel (eg. Stain-Free from BioRad). There are other possibilities of total protein fluorescent labeling that will be discussed. We will provide an example of this application.
    • On the need of antibodies and immunoblotting to decide on the best denaturing temperature for a specific protein or FP tag – we believe that the system could be set up for a protein of interest for which antibodies are available (as we did for Bmh1 or Hxk1), and once this is done, there is no need to do these controls anymore. We will mention this in the manuscript.
    • On the complexity of the native protein extraction protocol compared for example to alkaline lysis followed by TCA precipitation – indeed the protocol is a bit more time-consuming compared to the mentioned method, and we will mention this in the text. However, please note that people studying mammalian cells, for instance, often use this native protocol for total extracts so this is mostly a yeast-model issue. Yet, we will add this comment. Moreover, although the denatured fraction is FP- and temperature-dependent, even under the milder 30{degree sign}C conditions there is a detectable denatured fraction (Fig.s3b). This would seem to preclude the use of this approach for absolute protein quantification.

    True, but it depends a lot on the FP used. For instance, sfGFP is not denatured and could potentially be used for absolute quantification. We will comment in the text.

    Finally, any evidence that the denatured fraction would depend on the protein tagged with the FP?

    We will use several proteins used for point 1 but fused to the most sensitive FP, GFP-S65T, and do a western blot using anti-GFP antibodies to estimate the variation in native vs. denatured forms of the protein.

    Minor points:

    1. In the experiments designed to test the linearity and sensitivity of the approach, an alternative approach that would not result in dilution of cell extract is to mix wild type cell extract (no GFP fusion) with extract of the GPF-tagged strain in different ratios.

    Yes, this was an alternative but it seemed that dilution was easier to control than mixing two extracts.

    Define all acronyms at first appearance. For example, DTT and LDS on page 4.

    Thank you, we will address all acronyms in the text.

    Fig.4D: the colors chosen to represent EGFP and sfGFP data make them hard to tell apart. The same comment to Fig.S6.

    Agreed, we will change the figures accordingly.

    As the temperature steps are not uniform in Figures 4 and 5, it would be more informative to indicate the exact temperate above each lane (in addition/instead of the ramp cartoon).

    Agreed, we will change the figures accordingly.

    Regarding linearity, that HRP-based quantification is not linear is expected. A fairer comparison would be to use fluorescently labeled secondary antibodies. It is also puzzling that detection with signal amplification (HRP) is less sensitive than direct quantification of the fluorescence signal from the FP tag.

    We will do a sensitivity tets (dilutions) to compare IGF with HRP-based and fluorescent-based antibody-mediated detection.

    I appreciate the workflow Figure 10. But in my opinion it is trying to show too much (protocol, troubleshooting, calls to figure panels). Perhaps it could be made clearer by separating the protocol steps/settings from the optimization/troubleshooting tips.

    Thank you, we will work on this to make the workflow clearer.

    Some of the discussion of different fluorescent proteins, and expression levels of tagged proteins, could be confounded by the different linkers used in the tagging constructs.

    Thank you for this remark. Indeed, there are various linkers on these constructs and we don’t know to which extent they contribute to the effect on protein expression level. We will comment his in the text.

    Significance

    Could be a generally useful and simple approach for in-gel quantification using fluorescent protein tags.

    __ ____Thank you for your comments and overall assesment.__

    Reviewer #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 Campbell et al. Proc Natl Acad Sci USA. 2002 Jun 11, 99:7877-82). However, the authors did perform a very accurate and robust study to quantitatively assess the behavior of several FP fusion protein in SDS-PAGE. A thorough analysis of different conditions for a variety of FPs and target proteins was done; detailed protocols were developed. A surprisingly high sensitivity of FP detection (even superior to that of standard Western blotting) was demonstrated. Considering the simplicity of the proposed approach, it appears to be the method of choice for those working with FP fusion proteins.

    Thank you for this comment. Indeed we do not claim to discover that FP remain fluorescent in mild denaturing conditions, as presented in the text. We did our best to include original publications showing precedent for this and we missed Yanushevich et al. FEBS Lett. 2002 that we will add. However the Campbell paper is cited, precisely for the Supplementary figure 7 that the reviewer mentions.

    I have only minor, discretionary comments:

    1. It is known that under conditions of SDS-PAGE without heating, FPs retain not only fluorescence but also their oligomeric state. The same can be true for proteins of interest (POIs). If so, even for monomeric FPs, the POI-FP band can potentially migrate much slower than expected because of oligomerization of the POI.

    __Thank you for this suggestion. Our data in the manuscript already show that Bmh1 and Bmh2, which are tighlty associated 14-3-3 proteins, no longer intereact in these mild denaturation conditions. In the set of proteins that we will use to answer to Reviewer #1 (point 1), we will include proteins in large complexes to assess whether this can happen. __

    It might be useful to briefly discuss a possibility to use other types of fluorescent proteins (namely, Flavin-binding FPs, bacteriophytochrome-based FPs, bilirubin-binding FP UnaG) in the same way as proposed here. In particular, biliverdin-binding near-infrared FPs (IFP, iRFP, etc.) can be detected even after fully denaturing SDS-PAGE by zinc-induced orange fluorescence of proteins carrying covalently attached bilin chromophore (Berkelman TR, Lagarias JC. Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal Biochem. 1986, 156, 194-201; Stepanenko OV, Kuznetsova IM, Turoverov KK, Stepanenko OV. Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties. Int J Mol Sci. 2022, 23, 7347).

    __Agreed. ____We will extend the discussion to other fluorescent approaches to visualize proteins in gels and compare them. __

    Significance

    A simple method of specific visualization of fluorescent protein fusion bands on SDS-PAGE is proposed.

    Thank you for your comments and overall assesment.

    Reviewer #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 IGF is not only comparable but also superior to chemiluminescence. This is particularly significant given that chemiluminescence can suffer from issues such as a limited dynamic range and limitations in accurately quantifying very low or high-abundance proteins. The authors further demonstrate the utility of IGF in co-immunoprecipitation experiments and test whether the mild denaturing conditions are compatible with proteins from other organisms. Overall, the study is well-presented and is an asset to the scientific community. I have one major and some minor comments that, in my opinion, would improve this already informative paper: Major comment

    1. In all cases where there is signal quantification the authors should perform replicates to account for variability of the signal (in Fig 6, S6 and S7).

    __Agreed, we will perform triplicates for the indicated experiments. __

    Minor comments

    1. The study mainly focuses on soluble protein. While the authors have tested one plasma membrane protein, the study would benefit from including more membrane proteins from different environments (e.g., cell wall, nuclear envelope, mitochondrial). This would help determine if incubation at higher temperatures is necessary to properly solubilize these proteins, in which case the experiment would need adaptation.

    Thank you for this suggestion. __In the set of proteins that we will use to answer to Reviewer #1 (point 1), we will include proteins from various subcellular locations. __

    The authors show that when fluorescent proteins are partially denatured, their migration behavior changes. One cannot exclude that in some cases, the tagged proteins themselves might also be partially resistant to denaturing at the low temperatures used for IGF. This would lead to more than one fluorescent bands. In such cases one should be careful with interpretation, especially in the context of PTMs or isoforms. Could the authors briefly discuss this?

    Thank you for this comment. __We will discuss this in the text. __

    Based on Fig 4D and 5D, some fluorescent proteins seem to have a higher signal variability between replicates than others. It would be helpful to add this information next to the behavior of the proteins in different temperatures so it would be easier to choose the fluorescent protein for specific experiments.

    __Indeed, there are variations between experiments, but it is not clear whether this inherent to the FP considered or the experiment. We will look back at the data and modify the text accordingly if pertinent. __

    The sensitivity experiment (Figure 6) is convincing and important for IP conditions, where the total protein concentration of the sample is radically decreased. Could the authors additionally test if very low abundant proteins can be detected (without any dilution of the total protein content), and compare this to chemiluminescence? This could be done either by tagging some very low abundant proteins (for example a few hundred copies per cell) or diluting the lysate in wild-type lysate to artificially reduce their concentration while maintaining the overall protein load the same.

    __We have planned an experiment in which low abundant proteins will be tagged in response to reviewer 1 (point 1) which should address this point. __

    It would be useful to address the detection of very high molecular weight proteins - or proteins that are problematic in terms of transfer during western blotting.

    Again, in the experiment planned in ____response to reviewer 1 (point 1), proteins or various MW as well as membrane proteins will be studied, which should address this point. __ __

    Significance

    The authors already discuss the strengths and limitations of their approach. The main strength of IGF is that it does not require transfer of the proteins to a membrane and also does not rely on antibody binding and (potential) chemical reactions. In addition to the fact that this is time, cost, equipment, waste and expertise effective, the sensitivity and signal linearity of IGF seems to not only compare but outperforme western blotting. There are two main limitations. First, IGF relies on the resilience to denaturing of the chosen fluorescent protein that depends, according to the authors, at least on the temperature and overall protein concentration and pH. Second, IGF relied on tagging proteins with fluorescent proteins which might affect the stability or even function of the tagged protein. As the authors mention, these factors do not diminish the value of IGF, they highlight the need for appropriate controls.

    A potential development of the technique (not at the present study) could be the compatibility of IGF with different self-labelling proteins (Halo, Snap) and fluorescent dyes.

    We have conducted experiments in which we show the applicability of IGF in combination to SNAP-tagging, that we could show if needed.

    I think IGF will benefit a rather broad range of scientists. As already mentioned by the authors, there are different applications of IGF. From checking of clones when creating strains, to comparison of protein levels in different conditions and coIP experiments.

    Thank you for your comments and overall assesment.

    Cross reviews. Reviewer 1: I agree with the assessment by Reviewer #2. Considering the comment about potential oligomerization of a protein of interest, I stand by my point about testing the method with more proteins of interest. How extensive this testing should be or whether additional discussion of possible issues would suffice is a matter of opinion. It is clear from the manuscript in it's current form that the method works and that it has caveats.

    We believed that the experiments we have planned will clarify these points.

    Reviewer 2: In general, I agree with the points raised by Reviewer #1. However, in my opinion, there is already a large body of reliable experimental results in the manuscript that are worth publishing without a new round of extensive experiments.

    Reviewer 1: Fair enough, I don't insist on the experiments in my point 1.

    We think that this is an important point that will likely be a common question for readers so we will still do our best to provide data for this point.

  3. Review Commons

    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 IGF is not only comparable but also superior to chemiluminescence. This is particularly significant given that chemiluminescence can suffer from issues such as a limited dynamic range and limitations in accurately quantifying very low or high-abundance proteins. The authors further demonstrate the utility of IGF in co-immunoprecipitation experiments and test whether the mild denaturing conditions are compatible with proteins from other organisms.

    Overall, the study is well-presented and is an asset to the scientific community. I have one major and some minor comments that, in my opinion, would improve this already informative paper:

    Major comment

    1. In all cases where there is signal quantification the authors should perform replicates to account for variability of the signal (in Fig 6, S6 and S7). Minor comments
    2. The study mainly focuses on soluble protein. While the authors have tested one plasma membrane protein, the study would benefit from including more membrane proteins from different environments (e.g., cell wall, nuclear envelope, mitochondrial). This would help determine if incubation at higher temperatures is necessary to properly solubilize these proteins, in which case the experiment would need adaptation.
    3. The authors show that when fluorescent proteins are partially denatured, their migration behavior changes. One cannot exclude that in some cases, the tagged proteins themselves might also be partially resistant to denaturing at the low temperatures used for IGF. This would lead to more than one fluorescent bands. In such cases one should be careful with interpretation, especially in the context of PTMs or isoforms. Could the authors briefly discuss this?
    4. Based on Fig 4D and 5D, some fluorescent proteins seem to have a higher signal variability between replicates than others. It would be helpful to add this information next to the behavior of the proteins in different temperatures so it would be easier to choose the fluorescent protein for specific experiments
    5. The sensitivity experiment (Figure 6) is convincing and important for IP conditions, where the total protein concentration of the sample is radically decreased. Could the authors additionally test if very low abundant proteins can be detected (without any dilution of the total protein content), and compare this to chemiluminescence? This could be done either by tagging some very low abundant proteins (for example a few hundred copies per cell) or diluting the lysate in wild-type lysate to artificially reduce their concentration while maintaining the overall protein load the same.
    6. It would be useful to address the detection of very high molecular weight proteins - or proteins that are problematic in terms of transfer during western blotting.

    Significance

    The authors already discuss the strengths and limitations of their approach. The main strength of IGF is that it does not require transfer of the proteins to a membrane and also does not rely on antibody binding and (potential) chemical reactions. In addition to the fact that this is time, cost, equipment, waste and expertise effective, the sensitivity and signal linearity of IGF seems to not only compare but outperforme western blotting. There are two main limitations. First, IGF relies on the resilience to denaturing of the chosen fluorescent protein that depends, according to the authors, at least on the temperature and overall protein concentration and pH. Second, IGF relied on tagging proteins with fluorescent proteins which might affect the stability or even function of the tagged protein. As the authors mention, these factors do not diminish the value of IGF, they highlight the need for appropriate controls.

    A potential development of the technique (not at the present study) could be the compatibility of IGF with different self-labelling proteins (Halo, Snap) and fluorescent dyes.

    I think IGF will benefit a rather broad range of scientists. As already mentioned by the authors, there are different applications of IGF. From checking of clones when creating strains, to comparison of protein levels in different conditions and coIP experiments.

    Keywords/field of expertise: yeast genetics, organelle homeostasis, biochemistry, molecular biology, cell biology, fluorescence microscopy, functional proteomics, gut microbiology

  4. Review Commons

    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 Campbell et al. Proc Natl Acad Sci USA. 2002 Jun 11, 99:7877-82). However, the authors did perform a very accurate and robust study to quantitatively assess the behavior of several FP fusion protein in SDS-PAGE. A thorough analysis of different conditions for a variety of FPs and target proteins was done; detailed protocols were developed. A surprisingly high sensitivity of FP detection (even superior to that of standard Western blotting) was demonstrated. Considering the simplicity of the proposed approach, it appears to be the method of choice for those working with FP fusion proteins.

    I have only minor, discretionary comments:

    1. It is known that under conditions of SDS-PAGE without heating, FPs retain not only fluorescence but also their oligomeric state. The same can be true for proteins of interest (POIs). If so, even for monomeric FPs, the POI-FP band can potentially migrate much slower than expected because of oligomerization of the POI.
    2. It might be useful to briefly discuss a possibility to use other types of fluorescent proteins (namely, Flavin-binding FPs, bacteriophytochrome-based FPs, bilirubin-binding FP UnaG) in the same way as proposed here. In particular, biliverdin-binding near-infrared FPs (IFP, iRFP, etc.) can be detected even after fully denaturing SDS-PAGE by zinc-induced orange fluorescence of proteins carrying covalently attached bilin chromophore (Berkelman TR, Lagarias JC. Visualization of bilin-linked peptides and proteins in polyacrylamide gels. Anal Biochem. 1986, 156, 194-201; Stepanenko OV, Kuznetsova IM, Turoverov KK, Stepanenko OV. Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties. Int J Mol Sci. 2022, 23, 7347).

    Referee cross-commenting

    In general, I agree with the points raised by Reviewer #1. However, in my opinion, there is already a large body of reliable experimental results in the manuscript that are worth publishing without a new round of extensive experiments.

    Significance

    A simple method of specific visualization of fluorescent protein fusion bands on SDS-PAGE is proposed.

  5. Review Commons

    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 could be strengthened with more extensive benchmarking of the approach and further discussion of potential caveats as detailed below.

    Major points:

    1. Protein abundance in the original GFP library (and in other FP-tagged libraries constructed in the meanwhile) have been quantified using fluorescence (flow cytometry, microscopy, colony fluorescence) (Ho et al. 2018 10.1016/j.cels.2017.12.004, Weill et al. 2018 10.1038/s41592-018-0044-9, Meurer et al. 2018 10.1038/s41592-018-0045-8). This provides an opportunity to significantly strengthen the manuscript (where most of the test have been done using two abundant cytosolic proteins Bmh1 and Hxk1) if the authors could apply their approach to a representative fraction of the yeast proteome (sampling from such libraries FP-tagged proteins that differ in abundance, localization, membrane vs cytosolic/nuclear, subunits of large stable complexes vs proteins not part of complexes, etc.) and compare their quantification with previous relative abundance estimates. This information would also help future users in case protein-specific issues are identified.
    2. The authors discuss several drawbacks, including the change in apparent molecular weight compared to denatured proteins; differential recognition of folded vs denatured proteins by antibodies.

    Other potentials drawbacks should be discussed. For instance, the need of additional steps post-fluorescence imaging for signal normalization against a loading control; the need of antibodies and immunoblotting to decide on the best denaturing temperature for a specific protein or FP tag; complexity of the native protein extraction protocol compared for example to alkaline lysis followed by TCA precipitation (Knop et al. 1999 PMID: 10407276).

    Moreover, although the denatured fraction is FP- and temperature-dependent, even under the milder 30{degree sign}C conditions there is a detectable denatured fraction (Fig.s3b). This would seem to preclude the use of this approach for absolute protein quantification.

    Finally, any evidence that the denatured fraction would depend on the protein tagged with the FP?

    Minor points:

    1. In the experiments designed to test the linearity and sensitivity of the approach, an alternative approach that would not result in dilution of cell extract is to mix wild type cell extract (no GFP fusion) with extract of the GPF-tagged strain in different ratios.
    2. Define all acronyms at first appearance. For example, DTT and LDS on page 4.
    3. Fig.4D: the colors chosen to represent EGFP and sfGFP data make them hard to tell apart. The same comment to Fig.S6.
    4. As the temperature steps are not uniform in Figures 4 and 5, it would be more informative to indicate the exact temperate above each lane (in addition/instead of the ramp cartoon).
    5. Regarding linearity, that HRP-based quantification is not linear is expected. A fairer comparison would be to use fluorescently labeled secondary antibodies. It is also puzzling that detection with signal amplification (HRP) is less sensitive than direct quantification of the fluorescence signal from the FP tag.
    6. I appreciate the workflow Figure 10. But in my opinion it is trying to show too much (protocol, troubleshooting, calls to figure panels). Perhaps it could be made clearer by separating the protocol steps/settings from the optimization/troubleshooting tips.
    7. Some of the discussion of different fluorescent proteins, and expression levels of tagged proteins, could be confounded by the different linkers used in the tagging constructs.

    Referee Cross-commenting

    This session contains comments from all reviewers.

    Reviewer 1: I agree with the assessment by Reviewer #2. Considering the comment about potential oligomerization of a protein of interest, I stand by my point about testing the method with more proteins of interest. How extensive this testing should be or whether additional discussion of possible issues would suffice is a matter of opinion. It is clear from the manuscript in it's current form that the method works and that it has caveats.

    Reviewer 2: In general, I agree with the points raised by Reviewer #1. However, in my opinion, there is already a large body of reliable experimental results in the manuscript that are worth publishing without a new round of extensive experiments.

    Reviewer 1: Fair enough, I don't insist on the experiments in my point 1.

    Significance

    Could be a generally useful and simple approach for in-gel quantification using fluorescent protein tags.

  6. Version published to 10.1101/2024.05.31.594679v1 on bioRxiv