A guide to selecting high-performing antibodies for Prosaposin (UniProt ID: P07602) for use in western blot and immunoprecipitation

Introduction

Prosaposin, encoded by the PSAP gene, is a 527-amino acid protein, 70 kDa glycoprotein that serves as the precursor for four active saposins (A, B, C, and D). Following proteolytic cleavage, each saposin proteins plays a distinct role in the degradation of lysosomal sphingolipids.¹ In its full-length form, Prosaposin is secreted² and acts as a neurotrophic factor with protective effects.³ Loss of Prosaposin function results in a phenotype resembling type 2 Gaucher’s disease, an acute neuropathic disorder.⁴ Additionally, variants in the saposin D domain of PSAP gene have been directly linked to Parkinson’s disease.⁵

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.⁶ 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.⁶ Here we characterized nine commercial Prosaposin antibodies, selected and donated by participant antibody manufacturers, for use in western blot and immunoprecipitation, enabling biochemical and cellular assessment of Prosaposin 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 gateway⁷ and in Table 4 of this data note.⁶ Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway⁷ and in Table 4 of this data note.⁶

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

Western blot	Immunoprecipitation

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cell lines.^8,9 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 (Cancer Dependency Map Portal, RRID:SCR_017655) transcriptomics database to identify all 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.¹⁰ HeLa expresses the PSAP transcript at 8.4 log₂ TPM + 1. A PSAP KO cell line in HeLa was obtained from Abcam and was used for this study ( Table 1).

Prosaposin functions both as a secreted protein and intracellularly within the lysosome. Our aim was to detect both pools of Prosaposin. We screened all nine Prosaposin antibodies in western blot using HeLa WT and PSAP KO cells, analyzing both concentrated culture medium and cellular protein lysates. Samples were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed in parallel with the nine Prosaposin antibodies. While several antibodies detected Prosaposin in the culture medium ( Figure 1), intracellular Prosaposin was barely detectable in cell lysates (data not shown). This may be due to proteolytic cleavage of full-length Prosaposin within the cell, likely disrupting the epitopes required for antibody recognition.

Figure 1. Prosaposin antibody screening by western blot.

Culture media from HeLa WT and PSAP KO were collected, and 10 μg of protein were processed for western blot with the indicated Prosaposin antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO 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: ab166910** at 1/1000, ab308122** at 1/1000, NBP2–37422* at 1/200, AF8520 at 1/1000, GTX101064 at 1/500, 10801–1-AP at 1/1000, MA5–17159* at 1/200, MA5–38610* at 1/200, and MA5–48579* at 1/200. Predicted band size: 58.1 kDa. ** = recombinant antibody, * = monoclonal antibody.

We then assessed the capability of all nine antibodies to capture Prosaposin from HeLa WT culture medium using the immunoprecipitation technique, followed by western blot analysis. For the immunoblot step, a specific Prosaposin 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. Prosaposin antibody screening by immunoprecipitation.

Culture medium was collected from HeLa WT, and immunoprecipitation was performed for 1 h using 0.3 mg of protein and 2.0 μg of the indicated Prosaposin antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the anti-Prosaposin ab308122** diluted at 1/1000. The Ponceau stained transfers of each blot are shown. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate. ** = recombinant antibody, * = monoclonal antibody.

In conclusion, we have screened nine Prosaposin commercial antibodies by western blot and immunoprecipitation by comparing the signal produced by the antibodies in human HeLa WT and PSAP KO cells. To assist users in interpreting antibody performanyce, Table 4 outlines various scenarios in which antibodies may perform in these applications.¹⁰ High-quality and renewable antibodies that successfully detect Prosaposin was identified in both applications. Researchers who wish to study Prosaposin 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 Prosaposin 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 Prosaposin, only a brief overview of the protein’s function and its relevance in disease is provided. Prosaposin 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 ).⁶ Brief descriptions of the experimental setup used to carry out this study can be found below.