A guide to selecting high-performing antibodies for GCase (UniProt ID: P04062) for use in western blot, immunoprecipitation, and immunofluorescence

Introduction

Variants in GBA1, which encodes the lysosomal hydrolase glucocerebrosidase (GCase), represent one of the most common genetic risk factors for Parkinson’s disease.¹ Biallelic mutations in GBA1 are also causative of Gaucher’s disease, a lysosomal storage disorder.² GCase is responsible for the hydrolysis of glucosylceramide, and mutations in GBA1 can impair the enzyme’s structure, leading to reduced activity, stability, and protein levels.¹ The scientific community would benefit from specific and renewable GCase antibodies to investigate how disease-related mutations affect the protein’s biochemical and cellular characteristics.

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.³ 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.³ Here we characterized twenty-four commercial GCase 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 GCase 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 gateway⁴ and in Table 4 of this data note.³

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 cells.^5,6 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.⁷ The HAP1 expresses the GBA1 transcript at 3.5 log₂ TPM+1. A GBA1 KO HAP1 cell line was obtained from Horizon Discovery ( Table 1). Moreover, as seen on DepMap, the HAP1 does not carry mutations in the GBA1 that could affect antibody–epitope binding.

To screen all twenty-four by western blot, WT and GBA1 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with twenty-four GCase antibodies in parallel ( Figure 1). HeLa expresses the GBA1 transcript at 5.4 log₂ TPM+1, and a HeLa GBA1 KO is commercially available at Abcam. Protein expression of GCase was evaluated by western blot in the two cell types HAP1 and HeLa in addition to WT U-87 MG and RPE-1 ( Figure 2).

Figure 1. GCase antibody screening by western blot.

Lysates of HAP1 WT and GBA1 KO were prepared, and 30 μg of protein were processed for western blot with the indicated GCase 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. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilutions used: ab125065** at 1/100, ab128879** at 1/1000, ab309228** at 1/200, ab309229** at 1/200, ab55080* at 1/500, 2020A4849* at 1/1000, P1AM1914-001-28* at 1/1000, ABCD_AR148** 1/50 (1 μg/mL), ABCD_AR150** at 1/20, A19057** at 1/1000, NBP2-66871** at 1/500, MAB7410* at 1/500, 88162** at 1/100, GTX101267 at 1/100, GTX114073 at 1/1000, G4171 at 1/1000, 20622-1-AP at 1/100, 27972-1-AP at 1/1000, MA5-26589* at 1/100, MA5-26591* at 1/100, MA5-32591** at 1/1000, MA5-35236** at 1/1000, MA5-38382** at 1/1000, and MA5-52914** at 1/1000. Predicted band size: 59.7 kDa. **= recombinant antibody, *= monoclonal antibody.

Figure 2. GCase western blot on various cell lysates.

Lysates were prepared from WT and GBA1 KOs in HAP1, neural progenitor cells and HeLa as well as WT U-87 MG and RPE-1. 30 μg of protein were processed for western blot with anti-GCase A19057** diluted at 1/1000. Predicted band size: 59.7 kDa. **= recombinant antibody.

We then assessed the capability of all twenty-four antibodies to capture GCase from HAP1 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific GCase 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).

Figure 3. GCase antibody screening by immunoprecipitation on HAP1 lysates.

HAP1 lysates were prepared, and immunoprecipitation was performed using 1 mg of lysate and 2.0 μg of the indicated GCase antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the anti-GCase ab55080* diluted at 1/500 or A19057** at 1/1000. 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.

Based on the assessment of GCase expression in Figure 2, the highest expressing RPE-1 cells were selected for antibody screening in immunofluorescence. A KO cell line was generated by an academic partner in RPE-1 using CRISPR/Cas9. For immunofluorescence, twenty-two antibodies were screened using a mosaic strategy. First, RPE-1 WT and GBA1 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the GCase antibodies were evaluated. Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 4). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, and the images presented in Figure 4 are representative of this analysis.³

Figure 4. GCase antibody screening by immunofluorescence in RPE-1 cells.

RPE-1 WT and GBA1 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 GCase 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 merged 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 dilutions used: ab125065** at 1/50, ab128879** at 1/200, ab309228** at 1/500, ab309229** at 1/500, ab55080* at 1/1000, 2020A4849* at 1/500, P1AM1914-001-28* at 1/500, ABCD_AR148** 1/100, ABCD_AR150** at 1/100, A19057** at 1/500, NBP2-66871** at 1/1000, MAB7410* at 1/500, 88162** at 1/500, GTX101267 at 1/100, GTX114073 at 1/700, G4171 at 1/1000, 27972-1-AP at 1/300, MA5-26589* at 1/1000, MA5-26591* at 1/1000, MA5-32591** at 1/1000, MA5-35236** at 1/400, and MA5-38382** at 1/200. Bars = 10 μm. **= recombinant antibody, *= monoclonal antibody.

This study confirms the utility of the mouse monoclonal antibodies clones 1/17 and 1/23 for immunoprecipitation and immunofluorescence,⁸ and identifies rabbit recombinant antibodies suitable across multiple applications. The availability of both mouse- and rabbit-specific GCase antibodies enables multiplexing experiments and supports the development of antibody pairs for applications such as ELISA assays.

In conclusion, we have screened twenty-four GCase commercial antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human HAP1 and RPE-1 WT and GBA1 KO cells. To assist users in interpreting antibody performanyce, Table 4 outlines various scenarios in which antibodies may perform in all three applications.⁷ Several high-quality and renewable antibodies that successfully detect GCase were identified in all applications. Researchers who wish to study GCase 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 GCase 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 GCase, only a brief overview of the protein’s function and its relevance in disease is provided. GCase 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 ).³ Brief descriptions of the experimental setup used to carry out this study can be found below.