A guide to selecting high-performing antibodies for Stearoyl-CoA desaturase (SCD1) (UniProt ID: O00767) for use in western blot, immunoprecipitation, and immunofluorescence

Version Changes

Revised. Amendments from Version 1

This revised version incorporates edits made in response to reviewer comments, which have improved the clarity of the manuscript. A new Table 3 has been added to assist antibody users in interpreting the data presented. Additionally, a limitations section has been included to acknowledge the constraints inherent to this antibody guide.

Abstract

The enzyme stearoyl-CoA desaturase (SCD1) is a modulator of lipid metabolism by catalyzing the biosynthesis of mono-unsaturated fatty acids from saturated fatty acids. Understanding the specific mechanisms by which SCD1 plays in health and disease can provide novel insides in therapeutic targets, a process that would be facilitated by the availability of high-quality antibodies. Here we have characterized nine SCD1 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. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Introduction

Stearoyl-CoA desaturase (SCD1) is a membrane-bound enzyme which catalyzes the rate-limiting step in the conversion of saturated fatty acids into mono-unsaturated fatty acids. ^{1, 2} The regulation of SCD1 is physiologically important as maintaining a proper ratio of saturated to monounsaturated fatty acids is essential for membrane fluidity. Disruption to this ratio can lead to pathological conditions, including cardiovascular disease, obesity, non-insulin dependent diabetes mellitus, hypertension, neurological diseases, immune disorders and cancer. ^{2– 7}

SCD1 is of particular importance in Parkinson’s disease (PD) research as its inhibition has been found to rescue α-Synuclein cytotoxicity and inclusion formation, both hallmarks of PD progression. The neurotoxic mechanisms underlying PD progression are not yet clearly defined. ^{8– 10} Mechanistic studies would be facilitated with the availability of high-quality SCD1 antibodies.

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, ¹¹ and openly sharing the data. ^{12– 14} Here we evaluated the performance of nine commercial antibodies for SCD1 for use in western blot, immunoprecipitation, and immunofluorescence (also referred to as immunocytochemistry), enabling biochemical and cellular assessment of SCD1 properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available renewable antibodies against the corresponding protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). ¹⁵

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 Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway ¹⁶ and in Table 3 of this data note.

Table 3. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence.

Western blot	Immunoprecipitation	Immunofluorescence

This table has been reproduced with permission from Ayoubi et al., Elife, 2023. ¹²

Results and discussion

Our standard protocol involves comparing readouts from WT (wild type) and KO cells. ^{17, 18} The first step was to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log ₂ (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). The HeLa cell line expresses the SCD1 transcript at 6.7 log ₂ (TPM+1) RNA levels, which is above the average range of cancer cells analyzed, and does not carry mutations in the SCD gene that could affect antibody–epitope binding, as seen on DepMap. A SCD KO HeLa cells were obtained from Abcam ( Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Abcam	ab255448	CVCL_0030	HeLa	WT
Abcam	ab265220	CVCL_B2EP	HeLa	SCD KO

Limitations

Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available SCD1 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners.

Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in SCD1, only a brief overview of the protein’s function and its relevance in disease is provided. SCD1 experts are invited to analyze and interpret observed banding patterns in western blots and subcellular localization in immunofluorescence.

Thirdly, YCharOS experiments are not performed in replicates primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. Moreover, the same antibody clones are often donated by 2–3 manufacturers—such as the SCD1 antibodies ab19862 and MA5-27542 (clone CD.E10, cross-licensed between Abcam and Thermo Fisher)—effectively serving as replicates and enabling validation of test reproducibility. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In IF, the use of two different concentrations serves to evaluate antibody specificity and can aid in assessing assay reliability. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent data is performed subsequently to the antibody manufacturers internal validation process, therefore making our characterization process a repeat.

Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.

For western blot experiments, WT and SCD KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with nine antibodies in parallel ( Table 2, Figure 1).

Table 2. Summary of the SCD1 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
Abcam	ab19862 *	1057200-1	AB_445179	monoclonal	CD.E10	mouse	1.00	Wb, IP, IF
Abcam	ab236868 **	1007366-14	AB_2928123	recombinant mono	EPR21963	rabbit	0.61	Wb, IP, IF
Abcam	ab39969	1036585-6	AB_945374	polyclonal		rabbit	0.90	Wb
Aviva Systems Biology	ARP32797_T100	QC2226-43641	AB_841676	polyclonal		rabbit	1.00	Wb
Bio-Techne	AF7550	CGOP0121061	AB_3107036	polyclonal		sheep	0.20	Wb
Proteintech	28678-1-AP	00103543	AB_2923581	polyclonal		rabbit	0.40	Wb, IF
Thermo Fisher Scientific	MA5-27542 *	YH4004441A	AB_2723611	monoclonal	CD.E10	mouse	1.00	Wb, IP, IF
Thermo Fisher Scientific	PA5-75757	YJ4089139	AB_2719485	polyclonal		rabbit	1.00	Wb, IF
Thermo Fisher Scientific	PA5-95762	YJ4090059A	AB_2807564	polyclonal		rabbit	1.35	Wb, IF

Wb = western blot, IP = immunoprecipitation, IF = immunofluorescence.

^*Monoclonal antibody.

^**Recombinant antibody.

Figure 1. SCD1 antibody screening by western blot.

Lysates of HeLa WT and SCD KO were prepared, and 35 μg of protein were processed for western blot with the indicated SCD1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Tris-Glycine 4-20% gels were used. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody AF7550 which was titrated because the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab19862* at 1/1000, ab236868** at 1/1000, ab39969 at 1/1000, ARP32797_T100 at 1/1000, AF7550 at 1/200, 28678-1-AP at 1/1000, MA5-27542* at 1/1000, PA5-75757 at 1/200 and PA5-95762 at 1/1000. Predicted band size: 41.5 kDa *Monoclonal antibody, **Recombinant antibody.

We then assessed the capability of all nine antibodies to capture SCD1 from HeLa protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific SCD1 antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 2).

Figure 2. SCD1 antibody screening by immunoprecipitation.

HeLa lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated SCD1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the indicated SCD1 antibody. For western blot, MA5-27542* was used at 1/1000. Tris-Glycine 4-20% gels were used. The Ponceau stained transfers of each blot are shown. Predicted band size: 41.5 kDa. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate, HC= antibody heavy chain, LC= antibody light chain. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, nine antibodies were screened using a mosaic strategy. First, HeLa WT and SCD KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the SCD1 antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, and the images presented in Figure 3 are representative of this analysis. ¹⁵

Figure 3. SCD1 antibody screening by immunofluorescence.

HeLa WT and SCD KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio on coverslips. Cells were stained with the indicated SCD1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution. The rest of the antibodies were tested at 1 and 2 μg/mL and the final concentration was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilution used: ab19862* at 1/1000, ab236868** at 1/600, ab39969 at 1/150, ARP32797_T100 at 1/500, AF7550 at 1/200, 28678-1-AP at 1/400, MA5-27542* at 1/1000, PA5-75757 at 1/1000 and PA5-95762 at 1/1300. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened nine SCD1 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HeLa WT and SCD KO cells. High-quality and renewable antibodies that successfully detect SCD1 were identified in all applications. Researchers who wish to study SCD1 in a different species are encouraged to select high-quality antibodies based on the results presented and investigate the predicted species reactivity of the manufacturer before extending their research.

Method

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). ¹⁵

Antibodies and cell line used

Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID. ^{19, 20} All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free.

Funding Statement

This work was supported by a grant from the Michael J. Fox Parkinson’s Disease Research. It was also supported by the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

Underlying data

Zenodo: Antibody Characterization Report for SCD1, https://doi.org/10.5281/zenodo.13891494. ²¹

Zenodo: Dataset for the SCD1 antibody screening study, https://doi.org/10.5281/zenodo.14502183. ²²

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).