PONE-D-25-37026Validation of blue- and clear-native polyacrylamide gel electrophoresis protocols to characterize mitochondrial oxidative phosphorylation complexesPLOS ONE

Dear Dr. Taanman,

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Peng Gao, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Partly

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

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4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study presents a comprehensive validation of BN-PAGE and CN-PAGE protocols for characterizing OXPHOS complexes, with rigorous experimental design and robust supporting data. The following points should be addressed:

1.The authors should provide an explanation for the observed enhanced Complex IV staining in liver mitochondria relative to cultured cells. Given the liver's well-established high mitochondrial density and elevated metabolic activity, these inherent biological characteristics likely contribute to the differential staining outcomes. Clarification of this relationship would facilitate proper interpretation of the tissue-specific results.

2.For the technical limitations regarding Complex III/IV staining, additional discussion of potential underlying factors (e.g., enzyme stability or detergent compatibility) and attempted optimization strategies would strengthen the methodology section.

3.The wide range of protein loading quantities (5-30 μg) warrants clearer justification, especially whether this reflects biological sample differences or stain-specific sensitivity requirements.

4.The protocol's innovative aspects - particularly the streamlined extraction and enhanced Complex V detection - should be more prominently featured with direct comparisons to conventional methods.

5. Please discuss the similarities, differences, and innovations of this study in comparison to the referenced work (DOI: 10.3791/59294), particularly focusing on methodological advancements and biological implications.

6.Finally, a brief discussion of potential clinical applications, especially for mitochondrial disorder diagnostics, and sample-specific adaptation considerations would enhance the paper's translational impact.

Reviewer #2: This article validates a blue and transparent natural polyacrylamide gel electrophoresis (BN-PAGE and CN-PAGE) protocol for characterizing mitochondrial oxidative phosphorylation complexes. The study clearly describes the experimental protocol and provides sufficient experimental data to support its conclusions. This research has potential practical value as it simplifies the analysis process of mitochondrial oxidative phosphorylation complexes and enhances the sensitivity of certain enzyme activity staining.

Here are two suggestions:

1. The results section can be more concise and clear. For example, the description of the Western blot results can be simplified, with a focus on highlighting the key findings.

2. The discussion section can also explore the application prospects of this protocol in future research. For instance, it can discuss the potential applications of this protocol in diagnosing mitochondrial diseases and studying the effects of drugs on the OXPHOS system.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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Validation of blue- and clear-native polyacrylamide gel electrophoresis protocols to characterize mitochondrial oxidative phosphorylation complexes

PONE-D-25-37026R1

Dear Dr. Taanman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Peng Gao, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the protocol been described in sufficient detail?

To answer this question, please click the link to protocols.io in the Materials and Methods section of the manuscript (if a link has been provided) or consult the step-by-step protocol in the Supporting Information files.

The step-by-step protocol should contain sufficient detail for another researcher to be able to reproduce all experiments and analyses.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Does the protocol describe a validated method?

The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. If the manuscript contains new data, have the authors made this data fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the article presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have fully addressed and supplemented the previous reviewer comments, and conducted in-depth discussions on the key issues raised.

Reviewer #2: This article validates a blue and transparent natural polyacrylamide gel

electrophoresis (BN-PAGE and CN-PAGE) protocol for characterizing mitochondrial

oxidative phosphorylation complexes. The study clearly describes the experimental

protocol and provides sufficient experimental data to support its conclusions. This

research has potential practical value as it simplifies the analysis process of

mitochondrial oxidative phosphorylation complexes and enhances the sensitivity of

certain enzyme activity staining.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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