A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence

Introduction

Syntenin-1, encoded by SDCBP the gene, is a multifunctional PDZ-domain adaptor protein that integrates membrane dynamics with intracellular signaling. It organizes signalling complexes by binding to Syndecan and other partners, influencing cell membrane structure and trafficking.¹ Dysregulation of SDCBP expression has been increasingly associated with pathological phenotypes, most notably in cancer, where it promotes tumor cell survival, invasion, metastasis, and therapeutic resistance by modulating pathways such as Src, FAK, and Wnt/β-catenin.² RNA-seq data also reveals an increase of SDCBP levels in the brains of Alzheimer’s Disease patients when compared to controls.³

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, and openly sharing the data.⁴ 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.⁴ Here we characterized twelve commercial Syntenin-1 antibodies, selected and donated by participant antibody manufacturers, for use in western blot, immunoprecipitation, and immunofluorescence (also referred to as immunocytochemistry), enabling biochemical and cellular assessment of Syntenin-1 properties and function.

The authors do not engage in result analysis or offer explicit antibody recommendations. 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 gateway⁵ and in Table 4 of this data note.⁴

Table 4. Illustrations to assess antibody performance in all western blot, immunoprecipitation and immunofluorescence.

Western blot	Immunoprecipitation	Immunofluorescence

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cell lines.⁶ In the absence of commercially available KO cell lines, siRNA technology can be employed to knockdown (KD) the target gene.^7,8 To determine which cell line demonstrates high expression of SDCBP and thus be appropriate for KD, we examined the DepMap (Cancer Dependency Map Portal, RRID:SCR_017655) transcriptomics database to identify cell lines that express the target at levels greater than 2.5 log₂ (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off.⁹ As a result, HAP1 was identified, and a non-targeting control siRNA pool was used to treat HAP1 control (ctrl) cells, while SDCBP was KD using a pool of siRNA targeting this gene.

To screen the twelve antibodies by western blot, ctrl and SDCBP KD protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with all twelve Syntenin-1 antibodies in parallel ( Figure 1). To detect secreted Syntenin-1, six renewable recombinant antibodies were screened by western blot with HAP1 ctrl and SDCBP KD proteins from culture medium ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the six Syntenin-1 antibodies in parallel ( Figure 2).

Figure 1. Syntenin-1 antibody screening by western blot on lysate.

Lysates of HAP1 ctrl and SDCBP KD were prepared, and 30 μg of protein were processed for western blot with the indicated Syntenin-1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of ctrl and KD lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilutions used: ab133267** at 1/1000; A5497** at 1/500; ARP44535_T100 at 1/1000; ARP44537_P050 at 1/500; NBP3–26333** at 1/500; 27964** at 1/1000; GTX108391 at 1/500; GTX108470 at 1/500; GTX634154* at 1/500; MA5–34819** at 1/2000; MA5–35685** at 1/500; MA5–53244** at 1/1000. Predicted band size: 32.5 kDa. ** = recombinant antibody, * = monoclonal antibody.

Figure 2. Syntenin-1 antibody screening by western blot on culture medium.

Culture media from HAP1 ctrl and SDCBP KD were collected, and 30 μg of protein were processed for western blot with the indicated Syntenin-1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of ctrl and KD samples and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions used: ab133267** at 1/1000; A5497** at 1/500; 27964** at 1/1000; MA5–34819** at 1/2000; MA5–35685** at 1/500; MA5–53244** at 1/1000. Predicted band size: 32.5 kDa. ** = recombinant antibody, * = monoclonal antibody.

We then assessed the capability of all twelve antibodies to capture Syntenin-1 from HAP1 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific Syntenin-1 antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM) and the unbound fractions (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 3). The twelve antibodies were also tested for their ability to capture Syntenin-1 from HAP1 culture medium using the same immunoprecipitation technique ( Figure 4).

Figure 3. Syntenin-1 antibody screening by immunoprecipitation on lysate.

HAP1 WT lysates were prepared, and immunoprecipitation was performed for 1 h using 0.5 mg of lysate and 2.0 μg of the indicated Syntenin-1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the Syntenin-1 antibody MA5–35685** used at 1/500. The Ponceau stained transfers of each blot are shown. SM = 6% starting material; UB = 6% unbound fraction; IP = immunoprecipitate, ** = recombinant antibody, * = monoclonal antibody.

Figure 4. Syntenin-1 antibody screening by immunoprecipitation on culture medium.

Culture medium was collected from HAP1 WT, and immunoprecipitation was performed for 1 h using 0.35 mg of protein and 2.0 μg of the indicated Syntenin-1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the anti-Syntenin-1 antibody MA5–34819** diluted at 1/1000. The Ponceau stained transfers of each blot are shown. SM = 6% starting material; UB = 6% unbound fraction; IP = immunoprecipitate** = recombinant antibody, * = monoclonal antibody.

For immunofluorescence, the twelve antibodies were screened using a mosaic strategy. First, HAP1 ctrl and SDCBP KD cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Syntenin-1 antibodies were evaluated. Both ctrl and KD lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 5). Quantification of immunofluorescence intensity in hundreds of ctrl and KD cells was performed for each antibody tested, and the images presented in Figure 5 are representative of this analysis.⁴

Figure 5. Syntenin-1 antibody screening by immuofluorescence.

HAP1 ctrl and SDCBP KD cells were labelled with a green or a far-red fluorescent dye, respectively. Ctrl and KD cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated Syntenin-1 antibodies and with the corresponding fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (ctrl), red (antibody staining) and far-red (KD) channels was performed. Representative images of the blue and red (grayscale) channels are shown. Ctrl and KD 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 dilutions used: ab133267** at 1/500; A5497** at 1/800; ARP44535_T100 at 1/500; ARP44537_P050 at 1/500; NBP3–26333** at 1/100; 27964** at 1/100; GTX108391 at 1/500; GTX108470 at 1/500; GTX634154* at 1/500; MA5–34819** at 1000/; MA5–35685** at 1/800; MA5–53244** at 1/1000. Bars = 10 μm. ** = recombinant antibody, * = monoclonal antibody.

In conclusion, we have screened twelve Syntenin-1 commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HAP1 ctrl and SDCBP KD cells. Western blot and immunoprecipitation were performed in both lysate and conditioned medium. To assist users in interpreting antibody performance, Table 4 outlines various scenarios in which antibodies may perform in all three applications.⁹ High-quality and renewable antibodies that successfully detect Syntenin-1 were identified in all applications. Researchers who wish to study Syntenin-1 in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.

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 Syntenin-1 antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. We encourage readers to consult vendor documentation to identify the specific antigen each antibody is raised against, where such information is available.

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 Syntenin-1, only a brief overview of the protein’s function and its relevance in disease is provided. Syntenin-1 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. 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.

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 Protocols.io (protocols.io/view/a-consensus-platform-for-antibody-characterization ).⁴ Brief descriptions of the experimental setup used to carry out this study can be found below.