A guide to selecting high-performing antibodies for Netrin-1 (UniProt ID: O95631) for use in western blot and immunoprecipitation

Introduction

Netrin-1 encoded by the NTN1 gene belongs to the Netrins family of laminin-like proteins (the laminin superfamily) typically involved in cell and axon migration during embryogenesis.¹ Netrin-1 is a secreted protein that consists of 604 amino acids and contains an N-terminal laminin-like domain, three epidermal growth factor-like repeats responsible for binding to the receptors, and a C-terminal Netrin-like module which contributes to its secretion.^2,3 During embryonic development, Netrin-1 guides axonal growth, neuron migration and glial differentiation. In non-neural organs, Netrin-1 plays an important role in tissue morphogenesis of mammary glands, lungs and pancreas.⁴ It also acts as an apoptotic inhibitor during angiogenesis, impacting survival of the cells.⁵ Netrin-1 also modulates acute and chronic inflammation mechanisms to protect the cells⁶ and is dysregulated in multiple cancers, indicating its involvement in tumorigenesis regulation.³ Lastly, Parkinson’s disease patients exhibit reduced levels of Netrin-1 as compared to control, and the loss of this protein may be contributing to the disease progression.⁷

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.⁸ 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.⁸ Here we characterized fourteen commercial Netrin-1 antibodies, selected and donated by participant antibody manufacturers, for use in western blot and immunoprecipitation, enabling biochemical and cellular assessment of Netrin-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 gateway⁹ and in Table 3 of this data note.⁸ Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway⁹ and in Table 3 of this data note.⁸

Results and discussion

Our standard protocol involves comparing readouts from wild type (WT) and KO cell lines.^10,11 A commercially available NTN1 KO cell line in MCF7 was obtained from Abcam and was used for this study ( Table 1). DepMap (Cancer Dependency Map Portal, RRID:SCR_017655) is a transcriptomics database which helps 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 Since MCF7 expresses the NTN1 transcript at 1.6 log₂ TPM + 1, U-2 OS was selected as a second cell line to KO the corresponding NTN1 gene using CRISPR/Cas9 ( Table 1).

To detect secreted Netrin-1, all fourteen antibodies were first screened by western blot with the commercially accessible MCF7 WT and NTN1 KO proteins from culture medium ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with the fourteen Netrin-1 antibodies in parallel ( Figure 1). Next, the differrential expression of Netrin-1 was evaluated by western blot in culture media from various cell types ( Figure 2A) at different RNA levels ( Figure 2). U-2 OS WT and NTN1 KO were included in this cell panel and showed similar levels of secreted Netrin-1 in MCF7 and U-2 OS. Netrin-1 protein levels were also assessed in lysates from those same cell lines to explore the availability of the protein intracellularily prior to immunofluorescence. Intracellular Netrin-1 was detected at much lower levels than the secreted protein and required a higher amount of protein to be loaded onto the gel as well as significantly higher exposure times to attain detectable signal using two different antibodies in western blot ( Figure 2B). The immunofluorescence assay was thus dismissed in this data note.

Figure 1. Netrin-1 antibody screening by western blot on MCF7 culture medium.

Culture media from MCF7 WT and NTN1 KO were collected, and 10 μg of protein were processed for western blot with the indicated Netrin-1 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: ab126729** at 1/2000, NBP3–21882** at 1/1000, NBP3–32643** at 1/1000, AF6419 at 1/30, MAB1109* at 1/500, GTX133213 at 1/500, 20235–1-AP at 1/3000, PA5–98017 at 1/3000, IPIab00000283** at 1/100 (5 μg/mL), IPIab00000284** at 1/100, IPIab00000285** at 1/100, IPIab00000286** at 1/100, IPIab00000287** at 1/100, and IPIab00000288** at 1/100. Predicted band size: 67.7 kDa. ** = recombinant antibody, * = monoclonal antibody.

Figure 2. Netrin-1 western blot on culture media and lysates from various human cell lines.

A) Culture media were collected from WT and NTN1 KO MCF7, WT and NTN1 KO U-2 OS, WT U-87 MG, HeLa, HT-1080, SK-N-FI, A549, A-431, 293 T and OVCAR3, and 10 μg of protein were processed for western blot with anti-Netrin-1 ab126729** at 1/2000. B) Lysates were prepared from WT and NTN1 KO MCF7, WT U-2 OS, U-87 MG, HeLa, HT-1080, SK-N-FI, A549, A-431, 293 T and OVCAR3, and 30 μg of protein were processed for western blot with anti-Netrin-1 ab126729** at 1/2000 and NBP3–21882** at 1/1000. ** = recombinant antibody.

We then assessed the capability of all fourteen antibodies to capture Netrin-1 from MCF7 culture medium using the immunoprecipitation technique, followed by western blot analysis. For the immunoblot step, a specific Netrin-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).

Figure 3. Netrin-1 antibody screening by immunoprecipitation on MCF7 culture medium.

Culture medium was collected from MCF7, and immunoprecipitation was performed for 1 h using 0.15 mg of protein and 2.0 μg of the indicated Netrin-1 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for western blot with the anti-Netrin-1 ab126729** diluted at 1/2000. The Ponceau stained transfers of each blot are shown. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate; HC = antibody heavy chain. ** = recombinant antibody, * = monoclonal antibody.

In conclusion, we have screened fourteen Netrin-1 commercial antibodies by western blot and immunoprecipitation by comparing the signal produced by the antibodies in human MCF7 WT and NTN1 KO cells. To assist users in interpreting antibody performanyce, Table 3 outlines various scenarios in which antibodies may perform in these applications.¹² Several high-quality and renewable antibodies that successfully detect Netrin-1 were identified in both applications. Researchers who wish to study Netrin-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.

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

Western blot	Immunoprecipitation

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 Netrin-1 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 Netrin-1, only a brief overview of the protein’s function and its relevance in disease is provided. Netrin-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 ).⁸ Brief descriptions of the experimental setup used to carry out this study can be found below.