A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

Riham Ayoubi; Maryam Fotouhi; Donovan Worrall; Kathleen Southern; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium

doi:10.12688/f1000research.139867.2

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Data Note

Revised

A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

[version 2; peer review: 3 approved, 1 approved with reservations]

Previously titled: Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

Riham Ayoubi¹, Maryam Fotouhi¹, Donovan Worrall¹, [...] Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

Riham Ayoubi¹, Maryam Fotouhi¹, [...] Donovan Worrall¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

PUBLISHED 10 Apr 2024

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization

Maryam Fotouhi
Roles: Investigation, Validation

Donovan Worrall
Roles: Investigation, Visualization

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

Carl Laflamme
Roles: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Validation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Cell & Molecular Biology gateway.

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

Abstract

The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer’s Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence 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 P05067, APP, Amyloid-beta precursor protein, 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 knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-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 Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging under award number U54AG065187. The grant was from a Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (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 Ayoubi R 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: Ayoubi R, Fotouhi M, Worrall D et al. A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 3 approved, 1 approved with reservations]. F1000Research 2024, 12:956 (https://doi.org/10.12688/f1000research.139867.2) First published: 09 Aug 2023, 12:956 (https://doi.org/10.12688/f1000research.139867.1) Latest published: 10 Apr 2024, 12:956 (https://doi.org/10.12688/f1000research.139867.2)

Revised Amendments from Version 1

In this revised version of the article, modifications have been made to the language in the title, abstract, introduction and results&discussion section to clarify that this article, along with all Data Notes on the YCharOS gateway, does not provide result analysis nor recommendations for the antibodies tested. This article serves as a public resource, helping researchers select high quality antibodies for their studies related to amyloid-beta precursor protein. The ultimate objective is to reduce the use of poor-quality antibodies, thereby enhancing reproducibility of amyloid-beta precursor protein and Alzheimer disease related research while conserving both time and money.

References 6-8 and 21 have been added to help exemplify this reasoning as well as provide resources to help researchers interpret the results, if need be. The tables and figures remain the same as in Version 1.

See the authors' detailed response to the review by Amal Kaddoumi

Introduction

The amyloid-beta precursor protein, encoded by the APP gene, is a transmembrane protein with a single transmembrane domain, a large extracellular ectodomain and a short cytoplasmic tail.¹ Although ubiquitously expressed, amyloid-beta precursor protein is predominantly found on the surface of neurons, where it promotes neurite growth, neuronal adhesion and axonogenesis.²

Discovered and isolated in the 1980’s, amyloid-beta precursor protein has become a significant area of interest as its proteolytic cleavage can give rise to amyloid β-proteins (Aβ).¹^,³^,⁴ Aβ are small 40-42 amino acid-long peptides related to the pathogenesis of Alzheimer’s Disease as their accumulation can result in the formation of senile amyloid plaques.¹^,⁵ Further research is required to elucidate the pathway underlying the proteolytic processing of amyloid-beta precursor protein and in turn determine its potential as a diagnostic marker or therapeutic target to prevent degenerative brain progression. Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies. 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^–⁸

In this study, we compared the performance of eleven commercially-available antibodies for amyloid-beta precursor protein for use in Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of amyloid-beta precursor protein’s properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.⁹^–¹⁹ The first step was to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. 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). Commercially available HAP1 cells expressed the amyloid-beta precursor protein transcript at RNA levels above the average range of cancer cells analyzed. Parental and APP KO HAP1 cells were obtained from Horizon Discovery (Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Horizon Discovery	C631	CVCL_Y019	HAP1	WT
Horizon Discovery	HZGHC005368c010	CVCL_SD04	HAP1	APP KO

For Western Blot experiments, we resolved proteins from WT and APP KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).¹⁰^–¹⁹

Figure 1. Amyloid-beta precursor protein antibody screening by Western Blot.

Lysates of HAP1 (WT and APP KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated Amyloid-beta precursor protein 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. Exceptions were given for antibodies ab126732**, 29765** and 76600** which were all titrated to 1/500, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: ab126732** at 1/500, ab252814** at 1/500, ab252816** at 1/500, ARP33075 at 1/500, ARP34012 at 1/500, 29765** at 1/500, 76600** at 1/500, 25524-1-AP at 1/500, 13-0200* at 1/200, 14-9749-80* at 1/500, MA5-35187** at 1/500. Predicted band size: 87 kDa. *Monoclonal antibody, **Recombinant antibody.

For immunoprecipitation experiments, we used the antibodies to immunopurify amyloid-beta precursor protein from HAP1 cell extracts. The performance of each antibody was evaluated by detecting amyloid-beta precursor protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).¹⁰^–¹⁹

Figure 2. Amyloid-beta precursor protein antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated Amyloid-beta precursor protein antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated Amyloid-beta precursor protein antibodies. For Western Blot, MA5-35187** and ab252816** were used at 1/500. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate, *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, antibodies were screened using a mosaic strategy.²⁰ First, HAP1 WT and APP KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the eleven amyloid-beta precursor protein antibodies were evaluated by immunofluorescence. Cells were 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. The images presented in Figure 3 are representative of the results of this analysis.

Figure 3. Amyloid-beta precursor protein antibody screening by immunofluorescence.

HAP1 WT and APP 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 an optically clear flat-bottom. Cells were stained with the indicated Amyloid-beta precursor protein 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 the concentration was not indicated by the supplier, we tested antibodies at 1/100 and 1/500. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab126732** at 1/500, ab252814** at 1/500, ab252816** at 1/500, ARP33075 at 1/500, ARP34012 at 1/500, 29765** at 1/100, 76600** at 1/50, 25524-1-AP at 1/500, 13-0200* at 1/500, 14-9749-80* at 1/500, MA5-35187** at 1/1000. *Monoclonal antibody, **Recombinant antibody, Bars=10 μm.

In conclusion, we have screened eleven amyloid-beta precursor protein commercial antibodies by Western blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect amyloid-beta precursor under our standardized experimental conditions can be identified. In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. However, 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. An editorial by Biddle et al. provides valuable insights on how to interpret the antibody characterization data found in this article.²¹ The underlying data can be found on the Zenodo open access repository.²²^,²³

Methods

Antibodies

All amyloid-beta precursor protein antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.²⁴ Peroxidase-conjugated goat anti-mouse, anti-mouse and anti-rat antibodies are from Thermo Fisher Scientific (cat. number 62-6520, 65-6120 and 31470, respectively). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429).

Table 2. Summary of the amyloid-beta precursor protein antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μL)	Vendors recommended applications
Abcam	ab126732**	GR34478901	AB_11131727	recombinant-mono	EPR5118-34	rabbit	0.114	Wb
Abcam	ab252814**	GR34013051	AB_2925224¹	recombinant-mono	14D6	rat	1.154	Wb
Abcam	ab252816**	GR33342983	AB_2925223¹	recombinant-mono	2E9	rat	0.583	Wb, IP
Aviva Systems Biology	ARP33075	QC3561-90401	AB_2044934	polyclonal	-	rabbit	0.5	Wb
Aviva Systems Biology	ARP34012	QC47046-42571	AB_2044933	polyclonal	-	rabbit	0.5	Wb
Cell Signaling Technology	29765**	1	AB_2925221¹	recombinant-mono	E8B3O	rabbit	n/a	Wb, IP
Cell Signaling Technology	76600**	1	AB_2925222¹	recombinant-mono	E4H1U	rabbit	n/a	Wb, IP
Proteintech	25524-1-AP	51555	AB_2880118	polyclonal	-	rabbit	0.55	Wb, IF
Thermo Fisher Scientific	13-0200*	XC339604	AB_2532993	monoclonal	LN27	mouse	0.5	Wb, IP, IF
Thermo Fisher Scientific	14-9749-80*	2484403	AB_2572977	monoclonal	22C11	mouse	0.5	Wb, IF
Thermo Fisher Scientific	MA5-35187**	XH3670292	AB_2849091	recombinant-mono	ARC0465	rabbit	0.93	Wb, IF

* Monoclonal antibody.

** Recombinant antibody.

1 Refers to RRID recently added to the Antibody Registry (in May 2023), they will be available on the Registry website in coming weeks.

Cell culture

Both HAP1 WT and APP KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.²⁵ Cells were cultured in DMEM high glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin) and 100 μg/mL streptomycin (Wisent cat. number 450201).

Antibody screening by Western Blot

Western Blots were performed as described in our standard operating procedure.²⁶ HAP1 WT and APP KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used.

Western Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer (bio-Rad, cat. number 1610772), loaded in Laemmli loading sample buffer (Thermo Fisher Scientific, cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which was scanned to show together with individual Western blot. Blots were blocked with 5% milk for 1 h, and antibodies were incubated overnight at 4°C with 5% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 h at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL (Thermo Fisher Scientific, cat. number 32106) prior to detection with the iBright CL1500 Imaging System (Thermo Fisher Scientific, cat. number A44240).

Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure.²⁷ Antibody-bead conjugates were prepared by adding 2 μg or 20 μL of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer (Thermo Fisher Scientific, cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse and rat antibodies) (Thermo Fisher Scientific, cat. number 10002D and 10004D, respectively). Tubes were rocked for ~2 h at 4°C followed by two washes to remove unbound antibodies.

HAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked for 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 h at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western blot on precast midi 4-20% Tris-Glycine polyacrylamide gels.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.¹⁰^–²⁰ HAP1 WT and APP KO were labelled with a green and a deep red fluorescence dye, respectively (Thermo Fisher Scientific, cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. number D3571) fluorescent stain. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator at 37°C, 5% CO₂. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed three times with PBS. Cells were permeabilized in PBS with 0,1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095), 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary Amyloid-beta precursor protein antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 h at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20x/0.95 NA water objective lens and scientific CMOS camera (16-bit, 1.97 mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTrackerTM Green, Alexa fluor 555 and CellTrackerTM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTrackerTM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.²⁸ Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).

Data availability

Underlying data

Zenodo: Antibody Characterization Report for Amyloid-beta precursor protein, https://doi.org/10.5281/zenodo.7971926.²²

This project contains the following underlying data;

- Amyloid-beta precursor protein_APP_YCharOS report.pdf

Zenodo: Dataset for the Amyloid-beta precursor protein antibody screening study, https://doi.org/10.5281/zenodo.8140410.²³

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 knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. 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: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch, Kathleen Southern and Donovan Worrall.

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.

An earlier version of this article can be found on Zenodo (doi: 10.5281/zenodo.7971926)

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Version 2

VERSION 2 PUBLISHED 09 Aug 2023

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Methodology, Validation, Visualization

Maryam Fotouhi
Roles: Investigation, Validation

Donovan Worrall
Roles: Investigation, Visualization

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

Carl Laflamme
Roles: Conceptualization, Funding Acquisition, Methodology, Resources, Supervision, Validation

Competing interests

For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-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 Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging under award number U54AG065187. The grant was from a Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (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.

Article Versions (2)

version 2

Revised

Published: 10 Apr 2024, 12:956

https://doi.org/10.12688/f1000research.139867.2

version 1

Published: 09 Aug 2023, 12:956

https://doi.org/10.12688/f1000research.139867.1

© 2024 Ayoubi R 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.

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Ayoubi R, Fotouhi M, Worrall D et al. A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 3 approved, 1 approved with reservations]. F1000Research 2024, 12:956 (https://doi.org/10.12688/f1000research.139867.2)

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

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PUBLISHED 10 Apr 2024

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Reviewer Report 11 Oct 2024

Karen Bowman, University of Leicester, Leicester, England, UK

Approved

https://doi.org/10.5256/f1000research.164529.r326943

The study involved characterization of eleven commercial antibodies, from five different suppliers, for the amyloid-beta precursor protein (APP) for use in western blot, immunoprecipitation, and immunofluorescence. The study formed part of the wider YCharOS collaboration to characterize commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues. As a Data Note, it allows the direct comparison of the performance of commercially available antibodies to amyloid-beta precursor protein to aid scientists in the Alzheimer’s Disease community to choose the most appropriate antibody for their studies.
A committee of industry and academic representatives have endorsed the platform used, and the protocols used appear to be consistent with those in general use. The platform consisted of identification of a human cell line suitable for antibody characterization studies i.e., with adequate levels of the amyloid-beta precursor protein to generate a measurable signal. They found that the commercially available HAP1 cell line expressed the amyloid-beta precursor protein transcript at RNA levels above the average range of cancer cells, and therefore, it was a logical choice for their study. The matched isogenic knockout control cell line, APP KO HAP1 was used as an appropriate negative control. The final step was a series of antibody characterization procedures, limited to the most common research uses of antibodies. The interpretation of the results is left to the reader, with the study providing unbiased guidance. However, it gave some indication of the optimum dilutions of commercial antibodies. Two minor points to note. An ‘at a glance’ summary table encompassing the outcomes for each antibody would be useful. The IF figures are difficult to interpret using grayscale in this study, and it is not clear if any of the antibodies are suitable for IF. It might have been clearer to use the original colors.
In conclusion, the study will be a useful resource when choosing the most appropriate antibody for study of the amyloid-beta precursor protein.

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: Cell biology, DNA damage and repair, anti-cancer drug discovery.

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 01 Oct 2024

Jieya Shao, Washington University in St Louis, St. Louis, Missouri, USA

Approved

https://doi.org/10.5256/f1000research.164529.r326944

In this article the authors systematically compared the performance and specificity of eleven commercially available anti-amyloid-beta precursor protein (APP) antibodies using three different experimental approaches (Western blot, immunoprecipitation, and immunofluorescence staining). This study is part of a large collaborative initiative to characterize commercial antibodies for different human proteins and offer unbiased guidance to researchers who can select the best antibodies for their specific experimental objectives based on their own interpretations. As a Data Note, this paper successfully achieved its goal with rigorously collected high-quality data, and the results are self-explanatory and highly useful for the research community.

There are a few minor points to address. First, on page 5 under the methods-antibodies section, the authors mistakenly wrote anti-mouse secondary antibody twice one of which should be anti-rabbit. Second, on the same page under Western blot subsection, it may be helpful to briefly describe the method of protein normalization for equal loading. Third, on page 6 under Western blot subsection, the authors forgot to include the HAP1 APP KO cells in addition to WT.

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: cancer biology, DNA damage response, DNA replication stress

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 13 Apr 2024

Amal Kaddoumi, Auburn University, Auburn, Alabama, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.164529.r265022

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Version 1

VERSION 1

PUBLISHED 09 Aug 2023

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Reviewer Report 13 Feb 2024

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

Approved

https://doi.org/10.5256/f1000research.153179.r237210

This manuscript "Identification of high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence" fulfils the criteria for publication as a Data Note. The scientific topic - the immunological detection of the amyloid precursor protein APP in mammalian cells - is of current interest. On the one hand, this is based on research into the processes of neuronal development, homeostasis and interneuronal transmission and their connection to neurodegenerative diseases such as Alzheimer's disease. On the other hand, there are clear indications of increased expression of APP in malignant cells of various cancers.

In this context, it is very important to have appropriate, carefully characterized tools for the detection of the APP protein.

The manuscript contains a very impressive and well-designed overview and a head-to-head comparison of the performance of 11 commercially available antibodies from 5 different suppliers. It thus provides clear guidance for the selection of antibodies for a particular scientific or analytical question.

Two defined and well-characterized cell lines were used for the analysis, whose characteristics with regard to APP expression were clearly confirmed in the course of the work.

Minor Remark:
In this manuscript, the authors do not evaluate the individual antibodies in terms of their different performance in the results section. It would be helpful for the reader to be provided with such a summary or an overview of the results obtained for each antibody. This could be provided in the form of an additional table, or as a supplement in Table 2 in additional columns.

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: protein chemistry, antibody discovery, drug discovery, preclinical research, neurodegenerative diseases

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 24 Jan 2024

Amal Kaddoumi, Auburn University, Auburn, Alabama, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.153179.r237217

In this report, the authors compared eleven APP commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol using HAP1-APP expressing cells (WT) and HAP1-APP KO cells. This report could be useful if the authors elaborated more ... Continue reading

The authors should comment on the findings by clearly presenting their recommendations for which antibodies proved useful and for which assay. Also, they should comment on whether any of these antibodies could be used for the 3 assays, i.e., Western blot, immunoprecipitation, and immunofluorescence, or whether each assay requires one antibody but not the other.
It’s unclear how many times each antibody was tested for validation/consistency. Is one time run/test enough to conclude?
Each antibody specificity and species cross-reactivity should be reported.
The antibodies showed 2 bands. Please comment on these 2 bands.
Figure 3 presented binding in gray color; why is that? showing the images in their original staining colors (red, green, and blue for DAPI) would better clarify the binding. As presented, binding is not clear. Also, based on the results, which antibodies are recommended? As shown, the conclusions from Figure 3 are not clear.

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of methods and materials provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Neuropharmacology; Alzheimer's disease; CAA; blood-brain barrier

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, however I have significant reservations, as outlined above.

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Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that ... Continue reading
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

To your first comment regarding providing antibody recommendations for each application, we’d like to clarify that the aim of the YCharOS initiative is not to recommend antibodies based on their performance under our standardized protocol. Furthermore, given that this article is formatted as a Data Note, it does not require the results to be discussed nor concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. This study is to be used as a guide to help researchers, interested in studying amyloid-beta precursor protein or amyloid-beta proteins, select high-quality antibodies to enhance their experiments and prevent reproducibility and financial issues that occur when purchasing antibodies that don’t bind their target of interest properly. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies or providing recommendations for each application would be valid only under the precise experimental setup and cell line used.
That being said, we understand how the intention of our initiative may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure the authors goals are aligned and defined to all readers.

To your second comment, the antibodies are tested in each application once. The amount of antibody provided by our industry partners (usually 100 µl) is enough to perform each application once. Furthermore, all antibodies tested per human protein are evaluated side-by-side under the same experimental set-up providing more transparency and accuracy of results, even when subjected to the experiment only once.

For the same reasoning as in our reply to the first comment, the authors do not report antibody specificity or cross-reactivity. That being said, the following article written by Ayoubi et al., referenced in this article, describes the antibody performance criteria (Box 1) which may provide insights on the specificity of antibodies evaluated in this article (1)

In response to your fourth comment, according to the antibody manufacturer catalogs, all antibodies tested in this study are designed to target the amyloid-beta precursor protein. According to literature, amyloid-beta precursor protein undergoes post-translational proteolytic cleavage that gives rise to amyloid beta peptides via proteases (2-4). The appearance of two bands in Western blot and immunoprecipitation may be attributed to post-translation activity, although this was not confirmed and is outside the scope of this study. Further experiments by experts in the amyloid beta protein family is required, using the high-quality antibodies that can be identified in this study.

As described in the Figure legend of Figure 3, representative images are in gray due to the merged blue and red channel which generates a grayscale. The blue channel represents the nuclei stained with DAPI while the red channel represents the primary antibody staining. Therefore, the gray represents where the antibody is binding in the cell. Images of each channel can be found in the Dataset created for this study, in the Underlying data section.

Ayoubi, R., et al. (2023). "Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications." eLife 12: RP91645

Müller UC, Zheng H: Physiological functions of APP family proteins. Cold Spring Harb. Perspect. Med.2012;2(2):a006288. 22355794 10.1101/cshperspect.a006288

Glenner GG, Wong CW: Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984;120(3):885–890. 6375662 10.1016/S0006-291X(84)80190-

Kang J, Lemaire HG, Unterbeck A, et al.: The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733–736. 10.1038/325733a0
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

To your first comment regarding providing antibody recommendations for each application, we’d like to clarify that the aim of the YCharOS initiative is not to recommend antibodies based on their performance under our standardized protocol. Furthermore, given that this article is formatted as a Data Note, it does not require the results to be discussed nor concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. This study is to be used as a guide to help researchers, interested in studying amyloid-beta precursor protein or amyloid-beta proteins, select high-quality antibodies to enhance their experiments and prevent reproducibility and financial issues that occur when purchasing antibodies that don’t bind their target of interest properly. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies or providing recommendations for each application would be valid only under the precise experimental setup and cell line used.
That being said, we understand how the intention of our initiative may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure the authors goals are aligned and defined to all readers.

To your second comment, the antibodies are tested in each application once. The amount of antibody provided by our industry partners (usually 100 µl) is enough to perform each application once. Furthermore, all antibodies tested per human protein are evaluated side-by-side under the same experimental set-up providing more transparency and accuracy of results, even when subjected to the experiment only once.

For the same reasoning as in our reply to the first comment, the authors do not report antibody specificity or cross-reactivity. That being said, the following article written by Ayoubi et al., referenced in this article, describes the antibody performance criteria (Box 1) which may provide insights on the specificity of antibodies evaluated in this article (1)

In response to your fourth comment, according to the antibody manufacturer catalogs, all antibodies tested in this study are designed to target the amyloid-beta precursor protein. According to literature, amyloid-beta precursor protein undergoes post-translational proteolytic cleavage that gives rise to amyloid beta peptides via proteases (2-4). The appearance of two bands in Western blot and immunoprecipitation may be attributed to post-translation activity, although this was not confirmed and is outside the scope of this study. Further experiments by experts in the amyloid beta protein family is required, using the high-quality antibodies that can be identified in this study.

As described in the Figure legend of Figure 3, representative images are in gray due to the merged blue and red channel which generates a grayscale. The blue channel represents the nuclei stained with DAPI while the red channel represents the primary antibody staining. Therefore, the gray represents where the antibody is binding in the cell. Images of each channel can be found in the Dataset created for this study, in the Underlying data section.

Ayoubi, R., et al. (2023). "Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications." eLife 12: RP91645

Müller UC, Zheng H: Physiological functions of APP family proteins. Cold Spring Harb. Perspect. Med.2012;2(2):a006288. 22355794 10.1101/cshperspect.a006288

Glenner GG, Wong CW: Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984;120(3):885–890. 6375662 10.1016/S0006-291X(84)80190-

Kang J, Lemaire HG, Unterbeck A, et al.: The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733–736. 10.1038/325733a0
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Apr 2024

Author Response
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that ... Continue reading
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

To your first comment regarding providing antibody recommendations for each application, we’d like to clarify that the aim of the YCharOS initiative is not to recommend antibodies based on their performance under our standardized protocol. Furthermore, given that this article is formatted as a Data Note, it does not require the results to be discussed nor concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. This study is to be used as a guide to help researchers, interested in studying amyloid-beta precursor protein or amyloid-beta proteins, select high-quality antibodies to enhance their experiments and prevent reproducibility and financial issues that occur when purchasing antibodies that don’t bind their target of interest properly. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies or providing recommendations for each application would be valid only under the precise experimental setup and cell line used.
That being said, we understand how the intention of our initiative may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure the authors goals are aligned and defined to all readers.

To your second comment, the antibodies are tested in each application once. The amount of antibody provided by our industry partners (usually 100 µl) is enough to perform each application once. Furthermore, all antibodies tested per human protein are evaluated side-by-side under the same experimental set-up providing more transparency and accuracy of results, even when subjected to the experiment only once.

For the same reasoning as in our reply to the first comment, the authors do not report antibody specificity or cross-reactivity. That being said, the following article written by Ayoubi et al., referenced in this article, describes the antibody performance criteria (Box 1) which may provide insights on the specificity of antibodies evaluated in this article (1)

In response to your fourth comment, according to the antibody manufacturer catalogs, all antibodies tested in this study are designed to target the amyloid-beta precursor protein. According to literature, amyloid-beta precursor protein undergoes post-translational proteolytic cleavage that gives rise to amyloid beta peptides via proteases (2-4). The appearance of two bands in Western blot and immunoprecipitation may be attributed to post-translation activity, although this was not confirmed and is outside the scope of this study. Further experiments by experts in the amyloid beta protein family is required, using the high-quality antibodies that can be identified in this study.

As described in the Figure legend of Figure 3, representative images are in gray due to the merged blue and red channel which generates a grayscale. The blue channel represents the nuclei stained with DAPI while the red channel represents the primary antibody staining. Therefore, the gray represents where the antibody is binding in the cell. Images of each channel can be found in the Dataset created for this study, in the Underlying data section.

Ayoubi, R., et al. (2023). "Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications." eLife 12: RP91645

Müller UC, Zheng H: Physiological functions of APP family proteins. Cold Spring Harb. Perspect. Med.2012;2(2):a006288. 22355794 10.1101/cshperspect.a006288

Glenner GG, Wong CW: Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984;120(3):885–890. 6375662 10.1016/S0006-291X(84)80190-

Kang J, Lemaire HG, Unterbeck A, et al.: The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733–736. 10.1038/325733a0
Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

To your first comment regarding providing antibody recommendations for each application, we’d like to clarify that the aim of the YCharOS initiative is not to recommend antibodies based on their performance under our standardized protocol. Furthermore, given that this article is formatted as a Data Note, it does not require the results to be discussed nor concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. This study is to be used as a guide to help researchers, interested in studying amyloid-beta precursor protein or amyloid-beta proteins, select high-quality antibodies to enhance their experiments and prevent reproducibility and financial issues that occur when purchasing antibodies that don’t bind their target of interest properly. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies or providing recommendations for each application would be valid only under the precise experimental setup and cell line used.
That being said, we understand how the intention of our initiative may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure the authors goals are aligned and defined to all readers.

To your second comment, the antibodies are tested in each application once. The amount of antibody provided by our industry partners (usually 100 µl) is enough to perform each application once. Furthermore, all antibodies tested per human protein are evaluated side-by-side under the same experimental set-up providing more transparency and accuracy of results, even when subjected to the experiment only once.

For the same reasoning as in our reply to the first comment, the authors do not report antibody specificity or cross-reactivity. That being said, the following article written by Ayoubi et al., referenced in this article, describes the antibody performance criteria (Box 1) which may provide insights on the specificity of antibodies evaluated in this article (1)

In response to your fourth comment, according to the antibody manufacturer catalogs, all antibodies tested in this study are designed to target the amyloid-beta precursor protein. According to literature, amyloid-beta precursor protein undergoes post-translational proteolytic cleavage that gives rise to amyloid beta peptides via proteases (2-4). The appearance of two bands in Western blot and immunoprecipitation may be attributed to post-translation activity, although this was not confirmed and is outside the scope of this study. Further experiments by experts in the amyloid beta protein family is required, using the high-quality antibodies that can be identified in this study.

As described in the Figure legend of Figure 3, representative images are in gray due to the merged blue and red channel which generates a grayscale. The blue channel represents the nuclei stained with DAPI while the red channel represents the primary antibody staining. Therefore, the gray represents where the antibody is binding in the cell. Images of each channel can be found in the Dataset created for this study, in the Underlying data section.

Ayoubi, R., et al. (2023). "Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications." eLife 12: RP91645

Müller UC, Zheng H: Physiological functions of APP family proteins. Cold Spring Harb. Perspect. Med.2012;2(2):a006288. 22355794 10.1101/cshperspect.a006288

Glenner GG, Wong CW: Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984;120(3):885–890. 6375662 10.1016/S0006-291X(84)80190-

Kang J, Lemaire HG, Unterbeck A, et al.: The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733–736. 10.1038/325733a0
Competing Interests: No competing interests were disclosed. Close
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Version 2

VERSION 2 PUBLISHED 09 Aug 2023

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Amal Kaddoumi, Auburn University, Auburn, USA
Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany
Jieya Shao, Washington University in St Louis, St. Louis, USA
Karen Bowman, University of Leicester, Leicester, UK

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11 Oct 2024 | for Version 2

Karen Bowman, University of Leicester, Leicester, England, UK

2 Views Cite this report Responses(0)

Approved

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

Cell biology, DNA damage and repair, anti-cancer drug discovery.

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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5 Views

01 Oct 2024 | for Version 2

Jieya Shao, Washington University in St Louis, St. Louis, Missouri, USA

5 Views Cite this report Responses(0)

Approved

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

cancer biology, DNA damage response, DNA replication stress

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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13 Apr 2024 | for Version 2

Amal Kaddoumi, Auburn University, Auburn, Alabama, USA

16 Views Cite this report Responses(0)

Approved With Reservations

The authors did not address any of my previous comments. They modified the title to become a "Guide to selecting.... etc." and the abstract to include that this work is part of a larger study to "characterize commercially available antibodies etc.". Yet, guidance, characterization, discussions, and recommendations are not provided. This paper is descriptive and provides figures produced from 3 different assays for 11 tested antibodies without giving any guidance for selection or recommendations.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuropharmacology; Alzheimer's disease; CAA; blood-brain barrier

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16 Views

13 Feb 2024 | for Version 1

Jens-Ulrich Rahfeld, Fraunhofer Institute for Cell Therapy and Immunology, Department Molecular Drug Biochemistry and Therapy, Weinbergweg 22, 06120, Halle, Germany

16 Views Cite this report Responses(0)

Approved

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

protein chemistry, antibody discovery, drug discovery, preclinical research, neurodegenerative diseases

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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23 Views

24 Jan 2024 | for Version 1

Amal Kaddoumi, Auburn University, Auburn, Alabama, USA

23 Views Cite this report Responses(1)

Approved With Reservations

The authors should comment on the findings by clearly presenting their recommendations for which antibodies proved useful and for which assay. Also, they should comment on whether any of these antibodies could be used for the 3 assays, i.e., Western blot, immunoprecipitation, and immunofluorescence, or whether each assay requires one antibody but not the other.
It’s unclear how many times each antibody was tested for validation/consistency. Is one time run/test enough to conclude?
Each antibody specificity and species cross-reactivity should be reported.
The antibodies showed 2 bands. Please comment on these 2 bands.
Figure 3 presented binding in gray color; why is that? showing the images in their original staining colors (red, green, and blue for DAPI) would better clarify the binding. As presented, binding is not clear. Also, based on the results, which antibodies are recommended? As shown, the conclusions from Figure 3 are not clear.

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of methods and materials provided to allow replication by others?

No
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Neuropharmacology; Alzheimer's disease; CAA; blood-brain barrier

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Author Response

13 Apr 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Thank you Amal Kaddoumi for your comprehensive review of this study. A new version of this article has been submitted, to ensure transparency and prevent further misinterpretations. We trust that the refinements made meet your expectations, enhance comprehensibility and address any concerns you might have had. Please see our responses to your specific comments below.

To your first comment regarding providing antibody recommendations for each application, we’d like to clarify that the aim of the YCharOS initiative is not to recommend antibodies based on their performance under our standardized protocol. Furthermore, given that this article is formatted as a Data Note, it does not require the results to be discussed nor concluded. YCharOS is a public-private organization whose mission is to characterize antibodies for every human protein and deliver the research as a collective good for the scientific community. This study is to be used as a guide to help researchers, interested in studying amyloid-beta precursor protein or amyloid-beta proteins, select high-quality antibodies to enhance their experiments and prevent reproducibility and financial issues that occur when purchasing antibodies that don’t bind their target of interest properly. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies or providing recommendations for each application would be valid only under the precise experimental setup and cell line used.
That being said, we understand how the intention of our initiative may be misinterpreted. A new version has been submitted, to include modifications to the title, introduction and results&discussion section that ensure the authors goals are aligned and defined to all readers.

To your second comment, the antibodies are tested in each application once. The amount of antibody provided by our industry partners (usually 100 µl) is enough to perform each application once. Furthermore, all antibodies tested per human protein are evaluated side-by-side under the same experimental set-up providing more transparency and accuracy of results, even when subjected to the experiment only once.

For the same reasoning as in our reply to the first comment, the authors do not report antibody specificity or cross-reactivity. That being said, the following article written by Ayoubi et al., referenced in this article, describes the antibody performance criteria (Box 1) which may provide insights on the specificity of antibodies evaluated in this article (1)

In response to your fourth comment, according to the antibody manufacturer catalogs, all antibodies tested in this study are designed to target the amyloid-beta precursor protein. According to literature, amyloid-beta precursor protein undergoes post-translational proteolytic cleavage that gives rise to amyloid beta peptides via proteases (2-4). The appearance of two bands in Western blot and immunoprecipitation may be attributed to post-translation activity, although this was not confirmed and is outside the scope of this study. Further experiments by experts in the amyloid beta protein family is required, using the high-quality antibodies that can be identified in this study.

As described in the Figure legend of Figure 3, representative images are in gray due to the merged blue and red channel which generates a grayscale. The blue channel represents the nuclei stained with DAPI while the red channel represents the primary antibody staining. Therefore, the gray represents where the antibody is binding in the cell. Images of each channel can be found in the Dataset created for this study, in the Underlying data section.

Ayoubi, R., et al. (2023). "Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications." eLife 12: RP91645
Müller UC, Zheng H: Physiological functions of APP family proteins. Cold Spring Harb. Perspect. Med.2012;2(2):a006288. 22355794 10.1101/cshperspect.a006288
Glenner GG, Wong CW: Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 1984;120(3):885–890. 6375662 10.1016/S0006-291X(84)80190-
Kang J, Lemaire HG, Unterbeck A, et al.: The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987;325(6106):733–736. 10.1038/325733a0

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Figure 1. Amyloid-beta precursor protein antibody screening by Western Blot.

Figure 2. Amyloid-beta precursor protein antibody screening by immunoprecipitation.

Figure 3. Amyloid-beta precursor protein antibody screening by immunofluorescence.

Methods

Antibodies

Table 2. Summary of the amyloid-beta precursor protein antibodies tested.

Cell culture

Antibody screening by Western Blot

Antibody screening by immunoprecipitation

Antibody screening by immunofluorescence

Data availability

Underlying data

Acknowledgment

References

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