A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.


Introduction
Neurons communicate through regulated neurotransmitters release, a process controlled by calcium-dependent exocytosis of synaptic vesicles (SV). 1 Synaptotagmin-1, encoded by SYT1, is a transmembrane SV protein, with two tandem C2-domains (C2A and C2B).These domains sense and bind calcium ions, triggering a conformational change that induces SNARE-mediated fusion.[4][5] Mutations or dysregulation to SYT1 disrupts synaptic proteins and the synchronous release of neurotransmitters, causing damaging effects to the central nervous system, and contributes to neurodevelopmental conditions including epilepsy, 6 autism spectrum disorder 7 and neurodegenerative diseases such as Alzheimer's disease. 83][14] Here we evaluated the performance of thirteen commercial antibodies for Synaptotagmin-1 for use in western blot, immunoprecipitation, immunofluorescence, and flow cytometry enabling biochemical and cellular assessment of Synaptotagmin-1 properties and function.The platform for antibody characterization used to carry out this study was endorsed by a committee of industry academic representatives.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.The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 15e authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any conclusions remain relevant within the confines of the experimental setup and cell line used in this study.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 gateway. 16

Results and discussion
Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 17,18The first step is 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 transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655).HCT 116 cell line, which expresses the Synaptotagmin-1 transcript at 4.6 log 2 (TPM+1), was identified as a suitable cell line and was modified with CRISPR/Cas9 to KO the corresponding SYT1 gene (Table 1).
For western blot experiments, WT and SYT1 KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with thirteen Synaptotagmin-1 antibodies in parallel (Table 2, Figure 1).
We then assessed the capability of all thirteen antibodies to capture Synaptotagmin-1 from HCT 116 protein extracts using immunoprecipitation techniques, followed by western blot analysis.For the immunoblot step, a specific Synaptotagmin-1 antibody identified previously (refer to Figure 1) was selected.Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE (Figure 2).For immunofluorescence, thirteen antibodies were screened using a mosaic strategy.First, HCT 116 WT and SYT1 KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Synaptotagmin-1 antibodies were evaluated.Both WT and KO lines imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, 15 and the images presented in Figure 3 are representative of this analysis.
For flow cytometry, HCT 116 WT and SYT1 KO cells were labelled with distinct fluorescent dyes and combined at a 1:1 ratio.Both cell lines were fixed, permeabilized and blocked in the same tube prior to antibody staining to reduce bias.Nine Synaptotagmin-1 antibodies were then evaluated, with fluorescent intensity assessed using the Attune NxT flow cytometer.Antibody staining in both WT and KO line was then quantified using FlowJo software, with representative histograms presented (Figure 4).
In conclusion, we have screened thirteen Synaptotagmin-1 commercial antibodies by western blot, immunoprecipitation, immunofluorescence and flow cytometry by comparing the signal produced by the antibodies in human HCT 116 WT and SYT1 KO cells.Several high-quality and renewable antibodies that successfully detect Synaptotagmin-1 were identified in all applications.Researchers who wish to study Synaptotagmin-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.
The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. 19

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 Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 15tibodies and cell line used Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively.To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID. 20,21tibody screening by flow cytometry HCT 116 WT and SYT1 KO cells were detached, and three million cells were labelled with CellTracker green or violet fluorescent dyes, respectively (Thermo Fisher Scientific, cat.number C7025 and C10094).WT and KO cells were then centrifuged at 300 Â g, for 10 min and resuspended in 1% bovine serum albumin (BSA) (Wisent, cat.number 800-095) phosphate-buffered saline (PBS) (Wisent, cat.number 311-010).Cell populations were then combined at a 1:1 ratio, centrifuged and fixed on ice for 20 min using 800 μl of 4% PFA in PBS (Thermo Fisher Scientific, cat.number J61899).Following fixation, 1.2 ml of 1% BSA in PBS was added to the tube, vortexed and centrifuged at 600 Â g for 15 min at 4°C.Cells were permeabilized in PBS with 0.1% saponin for 10 min at room temperature, centrifuged at 600 Â g for 15 min at 4°C and then blocked with 5% goat serum (Gibco, cat.number 16210-064), 1% BSA in PBS for 30 min on ice.400,000 cells were aliquoted into individually labelled tubes, centrifuged 600 Â g for 15 min at 4°C and incubated in 150 μl of 1% BSA, 0.1% saponin PBS with primary Synaptotagmin-1 antibodies for 30 min on ice.500 μl of 1% BSA, 0.1% saponin PBS was added to each tube, vortexed and centrifuged at 600 Â g for 15 min at 4°C.Cells were then incubated the corresponding Multi-rAb CoraLite ® Plus 647 secondary antibodies (0.83 μg/ml) (Proteintech, cat.number RGAR005 and RGAM005) in 150 μl of 1% BSA, 0.1% saponin PBS for 30 min on ice.500 μl of 1% BSA, 0.1% saponin PBS was added to each tube, vortexed and centrifuged 600 Â g 15 min at 4°C.
Tubes were resuspended in 1 ml of 1% BSA in PBS and data was acquired using the Attune NxT flow cytometer.Data was analysed using FlowJo with the following gates.The cell population was gated on FSC-A vs SSC-A, within that gate single cells were selected by FSC-A vs FSC-H and then KO and WT cells were isolated by BL1-A vs VL1-A using a quadrat gate.Quantification of antibody staining was then observed in the RL1-A channel and histograms merged to demonstrate the staining intensity between the two populations.The figure was then assembled using Adobe Illustrator 2024.below.We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697 In their approach, they analyse the antibody performance in Western blot (WB), in immunoprecipitation (IP), in immunocytochemistry (staining of entire cells detected by immunofluorescence, IF) and even in flow cytometry (FC).
Although the objective of the authors are to present their approach as a method of picking a high performing antibody from the market, the data themselves are left without scientific assessment.I think this paper will benefit from an extra paragraph to discuss the quality of the individual data.For example, some antibodies show extra bands in WB, and it is not clear whether these bands would disappear upon further antibody dilution (without loss of specific signal), or that all the bands, the correct and the wrong ones, would equally fade by further antibody dilution.The target Synaptotagmin-1 is located in secretory vesicles and synaptic granules.Its location in the IF data is not very clear in many cases, located at the cellular membrane at best.A few sentences on the quality of the data would be desirable and put some extra scientific weight to the article.I would also like to read how the performance of the best antibodies compare across the different assays.It seems MAB4364 and 105011 are the best in FC, followed by 68043-1-Ig and 105008.But in IF the latter two outperform the former two.What is the authors' opinion on such inconsistency?Both assays are physically similar to one another and fundamentally different from WB and IP.All four antibodies perform extremely well in WB and IP.
The paper is extremely powerful and the first of its kind to rigorously assess the specificity and selectivity of commercial antibodies in such consistent way.I highly commend this paper for indexing, and I hope my remarks will be addressed in the final version.
Is the rationale for creating the dataset(s) clearly described?

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: Director and CEO of Aeonian Biotech, a consulting company helping to identify the best fit for purpose antibodies in the market.It also sells antibodies for research purposes.
Reviewer Expertise: Life-time career expertise on antibody characterisation and validation in all immunoassays.Both experience as an academic scientist, and as a commercial provider.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
In this manuscript, Biddle et al., present the characterization of 13 commercial antibodies targeting the neuronal synaptic vesicle protein Synaptotagmin-1.This is a protein essential for the fusion of synaptic vesicles and subsequent release of neurotransmitter into synaptic clefts, critical for neurotransmission in brain.Recent studies have identified novel loss of function mutations in patients suffering with neurodevelopmental delay underscoring the importance of developing tools for discovery purpose that accurately assess properties of Synaptotagmin-1 protein.
Antibodies are critical as binder tools to identify and characterize protein localization and function in health and disease, but are often presented as "working" in contexts outside those of the intended user.As such much frustration is gained and money lost in trying to use these tools from commercial vendors.
In the current manuscript, Biddle et al, in conjunction with the YCharos collective present the results of a detailed evaluation of commercially available Synaptotagmin-1 antibodies using a carefully established pipeline.After identifying an immortal cell line that expresses high levels of Synaptotagmin-1 endogenously, and obtaining a Synaptotagmin-1 KO version of the same cell line, the authors proceed to assess antibody performance in a variety of applications including western blot, immunoprecipitation, cell immunofluorescent labelling and flow cytometry.The results clearly identify set(s) of antibodies that work for each assay and seem to correctly identify Synaptotagmin-1, as well as many that fail.
The manuscript is clearly and concisely written.The methodologies utilized seem appropriate.Importantly, a standardized set of methods are employed for westerns, immunoprecipitation and immunofluorescent labeling of cultured cells, a necessary part of establishing a platform with wide utility for the scientific community.These protocols are available online.The flow cytometry method is adequately detailed in the current manuscript.The results are clearly presented and support the contention that open source characterization of antibodies (commercial or otherwise) using a generalized set of methods with WT and KO conditions can be extremely valuable to the broader scientific community and provide a highly valuable resource for guiding future work.Work of this nature has high value. Concerns: Figure 3: The authors state they quantitatively measures immunofluorescence content in "hundreds" of cells immunolabelled with the various antibodies yet this data was not presented.
As some of the antibodies do not appear to effectively label Synaptotagmin-1 its difficult to assess whether the labeling is affected in the KO cells.Quantitative numbers would help with assessment here.
Along same lines, the cell segmentation lines occlude assessment of the less effective labelers (eg mAb 30, 14558).Could they be limited to the DAPI panels?It's unfortunate the WT and KO cells are not from the same source.Its unclear how this could impact subtle changes in protein expression based on passage number, culture conditions etc that could impact some Abs but not others.It would be more ideal to generate the KO and WT from same exact source of cells, although this may be beyond the capabilities of the collective.Alternatively, running a control Ab, not expected to be impacted by the KO could be used as a control for cell line "matching".

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Neurodevelopment, plasticity, neurological disorders, antibody tool development and characterization I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure 2. Synaptotagmin-1 antibody screening by immunoprecipitation.HCT 116 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated Synaptotagmin-1 antibodies pre-coupled to Dynabeads protein A or protein G.The concentration of 14558** is unknown and therefore 10 μl of this antibody was tested.Samples were washed and processed for western blot with the indicated Synaptotagmin-1 antibody on a precast midi 4-20% Tris-Glycine polyacrylamide gel.For western blot, GTX637119** was used 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.*Monoclonal antibody; **Recombinant antibody.

Figure 4 .
Figure 4. Synaptotagmin-1 antibody screening by flow cytometry.HCT 116 WT and SYT1 KO cells were labelled with a green or violet, fluorescent dye, respectively.WT and KO cells were mixed in a 1:1 ratio, fixed in 4% PFA and permeabilized in 0.1% saponin.400,000 cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite ® Plus 647 secondary antibodies.Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the KO population (pink histogram, dashed line) compared to the WT cells (green histogram, solid line).Histograms with dotted lines represent secondary antibody-only controls in both WT and KO cells.All primary antibodies were diluted to 1 μg/ml except for 14558** which was used at 0.35 μg/ml (1/100) as the concentration was unknown.*Monoclonal antibody; **Recombinant antibody.

Table 1 .
Summary of the cell lines used.

Table 2 .
Summary of the Synaptotagmin-1 antibodies tested.
).Members of each group can be found below.NeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.ABIF consortium: Claire M. Brown and Joel Ryan.Thank you to the Structural Genomics Consortium, a registered charity (no.1097737), for your support on this project.The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no.OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no.875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.