Identification of high-performing antibodies for Superoxide dismutase [Cu-Zn] 1 (SOD1) for use in Western blot, immunoprecipitation, and immunofluorescence

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.


Introduction
Superoxide dismutase [Cu/Zn] 1 (SOD1) is an essential enzyme that protects the body against oxidative stress by acting as the first line of defense against reactive oxidative species. 1,2Largely cytosolic but also found in the mitochondrial intermembrane space, SOD1 is a 153 amino acid protein functioning as a homodimer to bind copper and zinc in order to carry out its role in scavenging free radicals. 3,4D1 was the first gene in which its mutations were identified in ALS over 30 years ago, predicting it to be a causative factor in motor neuron degeneration. 5A hallmark of SOD1-associated ALS is the misfolding and aggregation of SOD1 into neurotoxic species induced by gene mutations. 6The disease mechanism in which this occurs remains unknown. 6echanistic studies would be greatly facilitated with the availability of high-quality antibodies.
Here, we compared the performance of a range of commercially available antibodies for SOD1 and validated several antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of SOD1 properties and function.

Results and discussion
8][9] To identify a cell line that expressed adequate levels of SOD1 protein to provide sufficient signal to noise, we examined public proteomics databases, namely PaxDB 10 and DepMap. 11HeLa was identified as a suitable cell line and thus HeLa was modified with CRISPR/Cas9 to knockout the corresponding SOD1 gene (Table 1).
For Western blot experiments, we resolved proteins from WT and SOD1 KO cell extracts and probed them side-by-side with all antibodies in parallel 8,9 (Figure 1).SOD1 is an common essential gene 12 and the remaining SOD1 expression in the KO lysate could be detected with various antibodies.
For immunoprecipitation experiments, we used the antibodies to immunopurify SOD1 from HeLa cell extracts.The performance of each antibody was evaluated by detecting the SOD1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates 8,9 (Figure 2).For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 13In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).
In conclusion, we have screened SOD1 commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.Under our standardized experimental conditions, several high-quality antibodies were identified, however, the authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.

REVISED Amendments from Version 1
In this second version of the article, we have included a short paragraph to the Results and Discussion section to clarify that the authors do not score nor recommend antibodies and the reason why.We also provide the method used to quantify the HeLa lysates, which can be found in the Western blot methods.The title was also amended.
Any further responses from the reviewers can be found at the end of the article The underlying data can be found on Zenodo. 14,15thods Antibodies All SOD1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 16Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat.number 65-6120 and 62-6520).Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat.number A21424 and A21429).
CRISPR/Cas9 genome editing HeLa SOD1 KO clone was generated with low passage cells using an open-access protocol available on Zenodo.Two guide RNAs were used to introduce a STOP codon in the SOD1 gene (sequence guide 1: CCGTTGCAGTCCTCG-GAACC, sequence guide 2: GCGCGGGGGGACGAGCGGGT).Cell culture Both HeLa WT and SOD1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 17Cells were cultured in DMEM high-glucose (GE Healthcare cat.number SH30081.01) containing 10% fetal bovine serum (Wisent, cat.number 080450), 2 mM L-glutamate (Wisent cat.number 609065), 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat.number 450201).
Lysates were sonicated briefly and incubated for 30 min on ice.Lysates were spun at ~110,000 x g for 15 min at 4°C.To quantify the HeLa cell lysates, a BCA protein assay kit (Thermo Fisher Scientific, cat.number 23225) was used to measure protein concentration.Once the concentration was determined, equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western blot.BLUelf prestained protein ladder from GeneDireX (cat.number PM008-0500) was used.
Western blots were performed with large 8-16% polyacrylamide gels and transferred on nitrocellulose membranes.Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat.number BP103-10) which is scanned to show together with individual Western blot.Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat.number 800-095) in TBS with 0,1% Tween 20 (TBST) (Cell Signaling Technology, cat.number 9997).Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST.Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat.number 32106) prior to detection with the HyBlot CL autoradiography films (Denville, cat.number 1159T41).

Antibody screening by immunoprecipitation
Immunoprecipitation was performed as described in our standard operating procedure. 19Antibody-bead conjugates were prepared by 2.0 μg of antibody to 500 μL of phosphate-buffered saline (PBS) (Wisent, cat.number 311-010-CL) with 0,01% triton X-100 (Thermo Fisher Scientific, cat.number BP151-500) in a 1.5 mL microcentrifuge tube, together with 30 μL of protein A-(for rabbit antibodies) or protein G-(for mouse antibodies) Sepharose beads.Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.
HeLa WT were collected in HEPES lysis buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor.Lysates were rocked 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C.One mL aliquots at 0.5 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 hours at 4°C .The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of HEPES lysis buffer and processed for SDS-PAGE and Western blot on 8-16% polyacrylamide gels, as described above.Prot-A:HRP (MilliporeSigma, cat.number P8651) and VeriBlot for IP Detection Reagent HRP (Abcam, cat.number ab131366) were used as secondary detection systems for an experiment where a rabbit antibody was used for both immunoprecipitation and its corresponding Western blot.Similarly, anti-mouse IgG for IP HRP (Abcam, cat.number ab131368) was used for an experiment where a mouse antibody was used for immunoprecipitation and it's corresponding Western blot.

Antibody screening by immunofluorescence
Immunofluorescence was performed as described in our standard operating procedure. 8,9,13HeLa WT and SOD1 KO were labelled with a green and a far-red fluorescence dye, respectively.The fluorescent dyes used are from Thermo Fisher Scientific (cat.number C2925 and C34565).WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 o C, 5% CO 2 .Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat.number 140770-10ml) in PBS for 15 min at room temperature and then washed 3 times with PBS.Cells were permeabilized in PBS with 0,1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat.number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature.Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary SOD1 antibodies overnight at 4 °C.Cells were then washed 3 Â 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature.Cells were washed 3 Â 10 min with IF buffer and once with PBS.Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).
Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40x oil objective (NA = 1.40).Analysis was done using the Zen navigation software (Zeiss In general, the report was well-written and a few comments and recommendation are listed below: Please carefully check the whole text for typos, e.g."… an common …"; "An earlier version of this of this article…". 1.

2.
In the first paragraph of the Introduction, the authors mentioned that Sod1 is largely cytosolic but also found in the mitochondrial intermembrane space…".Sod1 is also present in the nucleus.Please add the citation below and clarify this point.Immunofluorescence, utilizing lysates from knockout cell lines and isogenic parental controls.The aim was to identify high-performing antibodies suitable for specific applications.While the authors have screened and compared the antibodies using different applications, there are some areas that require improvement, as outlined below: Quantification of HeLa lysates: The authors need to provide information on how the HeLa lysates were quantified.Adding details about the quantification method will enhance the reroducibility and accuracy of the results.

1.
Elaboration of the results section: The results section needs to be more detailed and informative.Currently, it lacks clarity regarding the outcomes for each technique and the identification of the best-suited antibodies for each application.

2.
Table of tested antibodies: To facilitate better understanding for readers, the authors should consider providing a table that lists the antibodies tested along with the techniques they were evaluated in.This table could highlight which antibodies performed best for each technique, making it easier for researchers to identify suitable antibodies for their experiments.

3.
By addressing these minor comments and enhancing the results section, the study's findings will become more accessible and beneficial to the scientific community.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?
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Table 1 .
Summary of the cell lines used.
).All cell images represent a single focal plane.Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).

appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Nishant N Vaikath Neurological
Disorder Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University, Doha, Doha, Qatar Thank you for the revised manuscript.I have no further comments to make.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 1
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
© 2023 N Vaikath N. Nishant N Vaikath Neurological Disorder Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University, Doha, Doha, Qatar In this study, Ayoubi et al, have characterized commercially available SOD1 antibodies for use in biochemical techniques, including Western Blotting, Immunoprecipitation, and