A guide to selecting high-performing antibodies for ASM (UniProt ID: P17405) for use in western blot and immunoprecipitation

Introduction

The sphingomyelin phosphodiesterase 1 (SMPD1) gene encodes acid sphingomyelinase (ASM), a lysosomal hydrolase that converts sphingomyelin to ceramide, a bioactive lipid critical for membrane integrity and cellular signaling.¹ Loss-of-function mutations in SMPD1 impair ASM activity, causing sphingomyelin accumulation and leading to Niemann-Pick disease types A and B, a lysosomal storage disorder characterized by neurodegeneration, neuroinflammation, and white matter abnormalities.² Beyond its canonical role in lysosomal degradation, ASM has been implicated in regulating membrane microdomain organization, autophagy, exosome release, and neuroimmune activation.^3,4 In Parkinson’s disease, reduced ASM activity and altered ceramide metabolism are associated with increased risk and earlier onset.⁵ Experimental models further show that ASM inhibition can modulate microglial activation, synaptic plasticity, and neuronal survival, underscoring the tight link between sphingolipid metabolism and brain homeostasis.⁶ These findings position SMPD1 as a molecular bridge between lysosomal function and neurodegenerative mechanisms, offering novel avenues for biomarker development and therapeutic intervention.

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.⁷ 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 antibodies against the corresponding protein.⁷ Here we characterized nine commercial ASM antibodies, selected and donated by participant antibody manufacturers, for use in western blot and immunoprecipitation, enabling biochemical and cellular assessment of ASM 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 gateway⁸ and in Table 4 of this data note.⁷

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

Western blot	Immunoprecipitation

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cell line.⁹ In the absence of a commercially available KO in a cell line background that expresses adequate levels of ASM, siRNA technology is employed to knockdown (KD) the target gene.^10,11 To determine which cell line demonstrates high expression of ASM and thus be appropriate for KD, the first step is to identify a cell line that expresses sufficient levels of a given protein to generate a measurable signal using antibodies. To this end, 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.¹² We selected the U-87 MG cell line that expresses the SMPD1 gene at 6.3 log₂ TPM + 1. A non-targeting control siRNA pool was used to treat U-87 MG control (ctrl) cells, while SMPD1 was KD using a pool of siRNA targeting this gene.

According to the UniProt database, ASM is predominantly secreted. To detect secreted ASM, all nine antibodies were first screened by western blot with U-87 MG ctrl and SMPD1 KD proteins from culture medium ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the nine ASM antibodies in parallel ( Figure 1A). Additionally, selected antibodies were then tested for intracellular ASM detection in protein extracts from U-87 MG control and SMPD1 knockdown cells ( Figure 1B). Although ASM was detectable in cell lysates, the signal required longer exposure times and higher protein loading compared to medium samples, indicating that most ASM is secreted. Based on these findings, the antibodies were not evaluated by immunofluorescence in this study.

Figure 1. ASM antibody screening by western blot.

A) Culture media from U-87 MG ctrl and SMPD1 KD were collected, and 10 μg of protein were processed for western blot with the indicated ASM 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 were chosen according to the recommendations of the antibody supplier. Antibody dilutions used: ab272729** at 1/1000, ab277243** at1/1000, ab277244** at 1/1000, ab315810 at 1/200, NBP2–45889* at 1/500, MAB5348* at 1/200, 3687 at 1/200, 14609–1-AP at 1/500 and MA5–26615* at 1/500. B) Lysates of U-87 MG ctrl and SMPD1 KD were prepared, and 30 μg of protein were processed for western blot with the selected ASM antibodies used at the same dilution as in A). Predicted band size: 69.9 kDa. ** = recombinant antibody, * = monoclonal antibody.

We then assessed the capability of the nine antibodies to capture ASM from U-87 MG culture medium using the immunoprecipitation technique, followed by western blot analysis. For the immunoblot step, a specific ASM 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 2).

Figure 2. ASM antibody screening by immunoprecipitation on culture medium.

Culture medium was collected from U-87 MG WT, and immunoprecipitation was performed for 1 h using 0.3 mg of protein and 2.0 μg of the indicated ASM antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the anti-ASM MAB5348* diluted at 1/500. The Ponceau stained transfers of each blot are shown. SM = 4% starting material; UB = 4% unbound fraction; IP = immunoprecipitate, HC = antibody heavy chain. ** = recombinant antibody, * = monoclonal antibody.

In conclusion, we have screened nine ASM commercial antibodies by western blot and immunoprecipitation by comparing the signal produced by the antibodies in human U-87 MG ctrl and SMPD1 KD cells. To assist users in interpreting antibody performanyce, Table 4 outlines various scenarios in which antibodies may perform in these applications.¹² Several high-quality and renewable antibodies that successfully detect ASM were identified in both applications. Researchers who wish to study ASM 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 ASM 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 ASM, only a brief overview of the protein’s function and its relevance in disease is provided. ASM 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 donated by 2–3 manufacturers (cross-licensed antibodies), effectively serving as replicates and enabling the 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.

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 ).⁷ Brief descriptions of the experimental setup used to carry out this study can be found below.

To knockdown SPMD1, U-87 MG cells were treated with the SMARTPool ON-TARGETplus Human SMPD1 siRNA from Horizon Discovery (cat. number L-006676-00-0005) for five days. U-87 MG control cells were treated with the ON-TARGETplus Non-targeting Control Pool (Horizon Discovery, cat. number. D-001810-10). Lipofectamine RNAiMAX (Thermo Fisher Scientific, cat. number 13778030) was used to transfect the siRNA following the manufacturer’s protocol.