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

Michael S. Biddle; Charles Alende; Maryam Fotouhi; Carolyn Jones; Riham Ayoubi; Kathleen Southern; Carl Laflamme; Harvinder Virk; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium

doi:10.12688/f1000research.154034.1

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

[version 1; peer review: 2 approved]

Michael S. Biddle¹, Charles Alende², Maryam Fotouhi², [...] Carolyn Jones¹, Riham Ayoubi², Kathleen Southern², Carl Laflamme ², Harvinder Virk¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

Michael S. Biddle¹, Charles Alende², [...] Maryam Fotouhi², Carolyn Jones¹, Riham Ayoubi², Kathleen Southern², Carl Laflamme ², Harvinder Virk¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

PUBLISHED 19 Jul 2024

Author details Author details

¹ NIHR BRC-Respiratory, University of Leicester, Leicester, England, UK
² Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Michael S. Biddle
Roles: Data Curation, Methodology, Validation, Writing – Original Draft Preparation

Charles Alende
Roles: Investigation, Visualization

Maryam Fotouhi
Roles: Investigation

Carolyn Jones
Roles: Investigation

Riham Ayoubi
Roles: Data Curation, Investigation, Methodology, Validation, Visualization

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision

Harvinder Virk
Roles: Data Curation, Funding Acquisition, Methodology, Supervision

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

Abstract

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.

Keywords

Uniprot ID P21579, SYT1, Synaptotagmin-1, antibody characterization, antibody validation, western blot, immunoprecipitation, immunofluorescence

Corresponding author: Carl Laflamme

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information: This work was supported in part by the Accelerating Medicines Partnership Program for Alzheimer’s disease (AMD-AD) project using a grant from the National Institute on Aging (grant no. U54AG065187). The Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Biddle MS et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Biddle MS, Alende C, Fotouhi M et al. A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry [version 1; peer review: 2 approved]. F1000Research 2024, 13:817 (https://doi.org/10.12688/f1000research.154034.1) First published: 19 Jul 2024, 13:817 (https://doi.org/10.12688/f1000research.154034.1) Latest published: 19 Jul 2024, 13:817 (https://doi.org/10.12688/f1000research.154034.1)

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. Sytnaptotagmin-1 thus couples Ca²⁺ influx to synchronous SV release of neurotransmitter to the presynaptic cleft.²^–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 disorder⁷ and neurodegenerative diseases such as Alzheimer’s disease.⁸ Synaptotagmin-1 is a focal research target, presenting as a potential biomarker for synaptic transmission in neurological health and disease.³^,⁵^,⁹^–¹¹

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.¹²^–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).¹⁵

The 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^,¹⁸ The 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 log₂ (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 log₂ (TPM+1), was identified as a suitable cell line and was modified with CRISPR/Cas9 to KO the corresponding SYT1 gene (Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Abcam	ab255451	CVCL_0291	HCT 116	WT
Montreal Neurological Institute	-	CVCL_B3P8	HCT 116	SYT1 KO

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).

Table 2. Summary of the Synaptotagmin-1 antibodies tested.

Company	Catalog number	Lot number (used in Wb, IP and IF)	Lot number (used in FC)	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
ABclonal	A0992	3561307005	-	AB_2757511	Polyclonal	-	rabbit	1.42	Wb, IF
Aviva Systems Biology	ARP59447	QC30410-220420	QC30410-220420	AB_10869736	Polyclonal	-	rabbit	0.50	Wb
Bio-Techne	MAB4364*	ZSJ0322021	ZSJ0322021	AB_2199304	Monoclonal	ASV48	mouse	0.50	Wb, IP
Cell Signaling Technology	14558**	1	1	AB_2798510	Recombinant-mono	D33B7	rabbit	n/a	Wb, IP
Developmental Studies Hybridoma Bank	mAB 30 (asv30)*	12/12/19	-	AB_2295002	Monoclonal	mAB 30 (asv30)	mouse	0.05	Wb, IP, IF
Developmental Studies Hybridoma Bank	mAb 48 (asv48)*	9/6/18	-	AB_2199314	Monoclonal	mAb 48 (asv 48)	mouse	0.33	Wb, IP, IF
GeneTex	GTX637119**	44886	44886	AB_3068350	Recombinant-mono	HL1626	rabbit	1.00	Wb, IF
GeneTex	GTX637252**	44886	44886	AB_3068349	Recombinant-mono	HL1654	rabbit	1.00	Wb, IF
Proteintech	14511-1-AP	00043462	00005811	AB_2199166	Polyclonal	-	rabbit	0.50	Wb, IP, IF
Proteintech	68043-1-Ig*	10024203	10024203	AB_2918785	Monoclonal	1H1H2	mouse	0.50	Wb
Synaptic Systems	105 008**	1-4	1-4	AB_2832236	Recombinant-mono	Rb41.1	rabbit	1.00	Wb, IP
Synaptic Systems	105 011*	1-64	1-64	AB_887832	Monoclonal	41.1	mouse	1.00	Wb, IP
Thermo Fisher Scientific	MA1-25568*	YJ4090338C	-	AB_795474	Monoclonal	ASV30	mouse	1.00	Wb, IP, IF

* Monoclonal antibody.

** Recombinant antibody.

Figure 1. Synaptotagmin-1 antibody screening by western blot.

Lysates of HCT 116 (WT and SYT1 KO) were prepared and 40 μg of protein were processed for western blot with the indicated Synaptotagmin-1 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 precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WPX42012BOX) to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 14511-1-AP and 68043-1-Ig* which were titrated as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: A0992 at 1/1000, ARP59447 at 1/1000, MAB4364* at 1/1000, 14558** at 1/1000, mAB 30 (asv30)* at 1/10, mAB 48 (asv8)* 1/10, GTX637119** at 1/1000, GTX637252** 1/1000, 14511-1-AP at 1/1000, 68043-1-Ig* 1/10000, 105 008** at 1/1000, 105 011* at 1/1000, MA1-25568* at 1/500. Predicted band size: 47.5 kDa. *Monoclonal antibody; **Recombinant antibody.

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).

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.

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.

Figure 3. Synaptotagmin-1 antibody screening by immunofluorescence.

HCT 116 WT and SYT1 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 in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated Synaptotagmin-1 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 dilution used: ARP59447 at 1/500, MAB4364* at 1/500, 14558** at 1/50, mAB 30 (asv30)* at 1/500, mAB 48 (asv8)* at 1/30, GTX637119** at 1/500, GTX637252** at 1/250, 14511-1-AP at 1/500, 6803-1-Ig* at 1/1000, 105 008** at 1/1000, 105 011 at 1/500, MA1-25568* at 1/1000. Bars = 10 μm. *Monoclonal antibody; **Recombinant antibody.

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).

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.

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).¹⁵

Antibodies 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^,²¹

Antibody 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 × 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 × 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 × 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 × 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 × 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 × 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.

Data availability

Underlying data

Zenodo: Dataset for the Synaptotagmin-1 antibody screening study, https://doi.org/10.5281/zenodo.12636746.¹⁹

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgment

We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). 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.

References

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Comments on this article Comments (0)

Version 1

VERSION 1 PUBLISHED 19 Jul 2024

Author details Author details

¹ NIHR BRC-Respiratory, University of Leicester, Leicester, England, UK
² Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Michael S. Biddle
Roles: Data Curation, Methodology, Validation, Writing – Original Draft Preparation

Charles Alende
Roles: Investigation, Visualization

Maryam Fotouhi
Roles: Investigation

Carolyn Jones
Roles: Investigation

Riham Ayoubi
Roles: Data Curation, Investigation, Methodology, Validation, Visualization

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision

Harvinder Virk
Roles: Data Curation, Funding Acquisition, Methodology, Supervision

Competing interests

For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and KO cell line providers. The partners provide antibodies and KO cell lines to the McPherson laboratory at no cost. These partners include: - Abcam- ABCD antibodies- ABclonal- Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information

This work was supported in part by the Accelerating Medicines Partnership Program for Alzheimer’s disease (AMD-AD) project using a grant from the National Institute on Aging (grant no. U54AG065187). The Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RA is supported by a Mitacs fellowship.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Biddle MS, Alende C, Fotouhi M et al. A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot ID P21579) for use in western blot, immunoprecipitation, immunofluorescence and flow cytometry [version 1; peer review: 2 approved]. F1000Research 2024, 13:817 (https://doi.org/10.12688/f1000research.154034.1)

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Open Peer Review

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Reviewer Report 20 Aug 2024

Jan Voskuil, Aeonian Biotech Netherlands, Leiden, The Netherlands

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https://doi.org/10.5256/f1000research.169011.r313882

The authors delivered an excellent approach to validate commercial antibodies by comparing their generated signals from a suitable cell line, proven to express the target gene sufficiently for antibody detection, with its cognate knock-out derivative cell line.

... Continue reading

The authors delivered an excellent approach to validate commercial antibodies by comparing their generated signals from a suitable cell line, proven to express the target gene sufficiently for antibody detection, with its cognate knock-out derivative cell line.

In their approach, they analyse the antibody performance in Western blot (WB), in immuno-precipitation (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?

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: 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.

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Reviewer Report 19 Aug 2024

Karl D Murray, University of California Davis School of Medicine, Davis, CA, USA

Approved

https://doi.org/10.5256/f1000research.169011.r310140

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 ... Continue reading

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.

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Version 1 19 Jul 24	read	read

Karl D Murray, University of California Davis School of Medicine, Davis, USA
Jan Voskuil, Aeonian Biotech Netherlands, Leiden, The Netherlands

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Reviewer Report

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20 Aug 2024 | for Version 1

Jan Voskuil, Aeonian Biotech Netherlands, Leiden, The Netherlands

9 Views Cite this report Responses(0)

Approved

The authors delivered an excellent approach to validate commercial antibodies by comparing their generated signals from a suitable cell line, proven to express the target gene sufficiently for antibody detection, with its cognate knock-out derivative cell line.

In their approach, they analyse the antibody performance in Western blot (WB), in immuno-precipitation (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?

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

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.

Respond to this report

Responses (0)

Back to all reports

Reviewer Report

4 Views

19 Aug 2024 | for Version 1

Karl D Murray, University of California Davis School of Medicine, Davis, CA, USA

4 Views Cite this report Responses(0)

Approved

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.

Respond to this report

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[16] 16. Biddle MS, Virk HS: YCharOS open antibody characterisation data: Lessons learned and progress made. F1000Res. 2023; 12: 1344. PubMed Abstract | Publisher Full Text | Free Full Text

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

Abstract

Keywords

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Table 2. Summary of the Synaptotagmin-1 antibodies tested.

Figure 1. Synaptotagmin-1 antibody screening by western blot.

Figure 2. Synaptotagmin-1 antibody screening by immunoprecipitation.

Figure 3. Synaptotagmin-1 antibody screening by immunofluorescence.

Figure 4. Synaptotagmin-1 antibody screening by flow cytometry.

Method

Antibodies and cell line used

Antibody screening by flow cytometry

Data availability

Underlying data

Acknowledgment

References

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