A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence

Maryam Fotouhi; Donovan Worrall; Riham Ayoubi; Kathleen Southern; Peter S. McPherson; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group; ABIF Consortium

doi:10.12688/f1000research.133645.2

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Revised

A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence

[version 2; peer review: 2 approved]

Maryam Fotouhi¹, Donovan Worrall¹, Riham Ayoubi¹, [...] Kathleen Southern¹, Peter S. McPherson¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF Consortium

Maryam Fotouhi¹, Donovan Worrall¹, [...] Riham Ayoubi¹, Kathleen Southern¹, Peter S. McPherson¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF Consortium

PUBLISHED 08 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

Maryam Fotouhi
Roles: Investigation

Donovan Worrall
Roles: Investigation, Visualization

Riham Ayoubi
Roles: Investigation, Methodology, Visualization

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

Peter S. McPherson
Roles: Conceptualization, Data Curation, Funding Acquisition, Resources, Supervision

Carl Laflamme
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

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

A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 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. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Keywords

Uniprot ID P31483, TIA1, RNA-binding protein TIA1, 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 ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP is a private-public partnership created by the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. 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). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds 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. 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 Fotouhi M 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: Fotouhi M, Worrall D, Ayoubi R et al. A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved]. F1000Research 2024, 12:745 (https://doi.org/10.12688/f1000research.133645.2) First published: 26 Jun 2023, 12:745 (https://doi.org/10.12688/f1000research.133645.1) Latest published: 08 Apr 2024, 12:745 (https://doi.org/10.12688/f1000research.133645.2)

Revised Amendments from Version 1

In this new version of the article, additional references have been included in the introduction to provide more extensive background on the role of TIA1 in various diseases. Additionally, in the Results & Discussion section, we clarify the YCharOS initiative as a public resource for the scientific community and encourage readers to use the data provided as a guide to selecting high-performing antibodies for their specific needs.

See the authors' detailed response to the review by Jozsef Gal

Introduction

T-cell intracellular antigen 1, or TIA1, is a cytotoxic granule-associated RNA-binding protein involved in regulating alternative pre-mRNA splicing and mRNA translation when bound to 3’ uridine-rich RNA sequences.¹^–⁴ Suppressing translation in environmentally stressed cells and promoting stress granule formation, TIA1 modulates cellular response to stress and inflammation.⁵^,⁶

Comparable to other RNA-binding proteins, disrupting the function of TIA1 can lead to various diseases including cancer,⁷^–⁹ autoimmune diseases¹⁰ and neurodegenerative disorders.¹¹^–¹⁴ Studies have demonstrated that mutations to the TIA1 gene may delay the disassembly of stress granule, resulting in insoluble and immobile stress granules, a clinical feature of ALS and FTD.⁶^,¹¹ Significant efforts are required to further elucidate the relationship between dysregulated RNA metabolism and ALS/FTD pathogenesis which may lead to novel therapeutic discoveries.¹¹^,¹⁵

Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies. Here, we compared the performance of a range of commercially-available antibodies for TIA1 and identified high-performing antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TIA1 properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.¹⁶^–²³ The first step was to identify a cell line(s) that expresses sufficient levels of TIA1 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 TIA1 transcript at RNA levels above the average range of cancer cells analyzed. Parental and TIA1 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	HZGHC003048C010	CVCL_TS30	HAP1	TIA1 KO

For Western Blot experiments, we resolved proteins from WT and TIA1 KO cell extracts and probed them side-by-side with all antibodies in parallel (Figure 1).¹⁷^–²³

Figure 1. TIA1 antibody screening by Western Blot.

Lysates of HAP1 (WT and TIA1 KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated TIA1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab140595** at 1/5000; ab263945** at 1/1000; A6237 at 1/1000; ARP40979 at 1/500; ARP40981 at 1/200; NBP2-53336* at 1/1000; NBP2-67203** at 1/1000; NBP3-13791** at 1/500; 86050** at 1/1000; GTX33545 at 1/500; MA5-26474* at 1/2000; and MA5-32615** at 1/500. Predicted band size: 43 kDa. *=monoclonal antibody, **=recombinant antibody.

For immunoprecipitation experiments, we used the antibodies to immunopurify TIA1 from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the TIA1 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).¹⁷^–²³

Figure 2. TIA1 antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 1.0 μg of the indicated TIA1 antibodies pre-coupled to Dynabeads protein G or protein A. (A) Ability of the antibodies to capture TIA1 was assessed by comparing the level of protein available in the starting material to the level remaining in the unbound fraction. (B) The immunoprecipitates for antibodies which could immunocapture TIA1 in (A) are shown. For Western Blot, 86050** was used at 1/1000 in A) and B). The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *=monoclonal antibody, **=recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy.²⁴ In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).

Figure 3. TIA1 antibody screening by immunofluorescence.

HAP1 WT and TIA1 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 TIA1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) 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. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given to antibodies ab140595**, A6237, NBP2-67203**, and GTX33545, which were titrated to their respective concentrations found bellow, as the signals were too weak when following the suppliers' recommendations. When the concentrations were not indicated by the supplier, which was the case for antibodies ab263945**, ARP40979, ARP40981, and NBP3-13791** we tested antibodies at 1/300, 1/500, 1/500 and 1/200, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab140595** at 1/600; ab263945** at 1/300; A6237 at 1/1000; ARP40979 at 1/500; ARP40981 at 1/500; NBP2-53336* at 1/100; NBP2-67203** at 1/1000; NBP3-13791** at 1/200; 86050** at 1/100; GTX33545 at 1/800; MA5-26474* at 1/1000; and MA5-32615** at 1/100. Bars = 10 μm. *=monoclonal antibody, **=recombinant antibody.

In conclusion, we have screened TIA1 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect TIA1 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. Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway.²⁵ The underlying data can be found on Zenodo open access repository.²⁶^,²⁷

Methods

Antibodies

All tested TIA1 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.²⁸ Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Table 2. Summary of the TIA1 antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications
Abcam	ab140595**	GR223312-17	AB_2687963	recombinant-mono	EPR9304	rabbit	0.665	Wb, IP, IF
Abcam	ab263945**	GR3297000-3	AB_2885132	recombinant-mono	EPR22999-80	rabbit	0.599	Wb, IP
Abclonal	A6237	18980101	AB_2766845	polyclonal	-	rabbit	2.920	Wb, IF
Aviva Systems Biology	ARP40979	QC10247-90408	AB_2048416	polyclonal	-	rabbit	0.500	Wb
Aviva Systems Biology	ARP40981	QC10249-41152	AB_938457	polyclonal	-	rabbit	0.500	Wb, IP
Bio-Techne	NBP2-53336*	7072-1p210821	AB_2885158	monoclonal	TIA1/1313	mouse	0.200	Wb, IF
Bio-Techne	NBP2-67203**	H00823	AB_2927744¹	recombinant-mono	JM42-11	rabbit	1.000	Wb, IP, IF
Bio-Techne	NBP3-13791**	7072-2P210904	AB_2927743¹	recombinant-mono	TIA1/1352R	rabbit	0.200	other
Cell Signaling Technology	86050**	1	AB_2800070	recombinant-mono	D1Q3K	rabbit	0.002	Wb, IP
GeneTex	GTX33545	822104511	AB_2887719	polyclonal	-	rabbit	0.820	Wb, IF
Thermo Fisher Scientific	MA5-26474*	VL3152368	AB_2725518	monoclonal	OTI1D7	mouse	1.000	Wb, IF
Thermo Fisher Scientific	MA5-32615**	VL3152613	AB_2809892	recombinant-mono	JM42-11	rabbit	1.000	Wb, IP, IF

* = monoclonal antibody.

** = recombinant antibody.

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

Cell culture

Both HAP1 WT and TIA1 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.²⁹ 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-glutamine (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.³⁰ HAP1 WT and TIA1 KO were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 89901) supplemented with 1x protease inhibitor cocktail (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 x 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 from GeneDireX (cat. number PM008-0500) was used.

Western Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from Bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred onto nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095) 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 hr at room temperature followed by three washes with TBST. Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with the HyBlot CL autoradiography films from Denville (cat. number 1159T41).

Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure.³¹ Antibody-bead conjugates were prepared by adding 1 μg of antibody to 500 μL of Pierce IP Lysis Buffer from 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 antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 hr 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 1x protease inhibitor (Millipore Sigma, cat. number P8340). Lysates were rocked 30 min at 4°C and spun at 110,000 x 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 ~1 hr at 4°C. The antibody-bead conjugate can elute the protein of interest if the tested antibodies can successfully bind their antigen. The unbound fractions were collected and removed while the beads were subsequently washed three times with 1.0 mL of or IP lysis buffer and processed for SDS-PAGE and Western Blot on a precast midi 4-20% Tris-Glycine polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.3 μg/mL for experiments where rabbit antibodies are used for both immunoprecipitation and its corresponding Western Blot.

Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure.¹⁷^–²⁴ HAP1 WT and TIA1 KO were labelled with a CellTracker^TM green (Thermo Fisher Scientific, cat. number C2925) or CellTracker^TM deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. WT and KO cells were plated in 96-well plate with optically clear flat-bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37^oC, 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 3 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 containing 5% BSA, 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 TIA1 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 hr at room temperature with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain, used to label the nuclei. 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/0.45 NA air objective lens and scientific CMOS camera (16-bit, 1.97mm 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, CellTracker^TM green, Alexa fluor 555 and CellTracker^TM deep red, respectively. Images had pixel sizes of 0.68 x 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 CellTracker^TM 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.³² 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 TIA1, https://doi.org/10.5281/zenodo.7671718.²⁶

Zenodo: Dataset for the TIA1 antibody screening study, https://doi.org/10.5281/zenodo.7796012.²⁷

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.

An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.7671718)

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Version 2

VERSION 2 PUBLISHED 26 Jun 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

Maryam Fotouhi
Roles: Investigation

Donovan Worrall
Roles: Investigation, Visualization

Riham Ayoubi
Roles: Investigation, Methodology, Visualization

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

Peter S. McPherson
Roles: Conceptualization, Data Curation, Funding Acquisition, Resources, Supervision

Carl Laflamme
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

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 ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP is a private-public partnership created by the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of Canada. 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). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds 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. 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)

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https://doi.org/10.12688/f1000research.133645.2

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© 2024 Fotouhi M 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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Fotouhi M, Worrall D, Ayoubi R et al. A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved]. F1000Research 2024, 12:745 (https://doi.org/10.12688/f1000research.133645.2)

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Reviewer Report 17 Apr 2024

Jozsef Gal, Radiation Medicine, University of Kentucky, Lexington, Kentucky, USA; Markey Cancer Center, University of Kentucky, Lexington, Kentucky, USA

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https://doi.org/10.5256/f1000research.164350.r264019

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Reviewer Report 20 Mar 2024

Jozsef Gal, Radiation Medicine, University of Kentucky, Lexington, Kentucky, USA; Markey Cancer Center, University of Kentucky, Lexington, Kentucky, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.146653.r204755

This article by Fotouhi et al. describes the validation of 12 commercially available anti-TIA1 antibodies in immunoblotting, immunoprecipitation and immunofluorescence studies. The manuscript is well written. This is a very useful contribution to the field. It will certainly help researchers ... Continue reading

The HAP1 cell line is of human origin. A lot of research employs murine models and cell lines. It would be very useful if the Authors could perform immunoblotting of a lysate from a murine cell line. I would recommend N2A/Neuro-2A cells. In my opinion, the antibodies that performed the best in HAP1 immunoblotting (ab140595, ab263945, NBP2-67203, 86050) would be sufficient.
The Authors present their findings in a rather raw format without much interpretation. It would be great if the Authors would summarize their findings after each technique. It would also be very useful to provide a summary paragraph about which antibodies the Authors consider overall the best.

Minor comments:

The term “Western blot” is kind of colloquial. I recommend the term “immunoblotting”, but I would like to leave it up to the Authors.
TIA-1 has a close homolog in human cells, TIAR. To me, one of the most valuable contributions of this work is the ability to see which tested antibodies may cross-react with TIAR. Based on the immunoblotting image, A6237, GTX33545, and MA5-26474 may cross-react with TIAR. I think that it would be useful if the Authors would touch on this issue.
In the Introduction: “... disrupting the function of TIA1 can lead to various diseases including cancer, autoimmune diseases and neurodegenerative disorders” – I recommend including further references in this paragraph for cancer and autoimmune diseases.
Could you, please, check the molecular weight marker bands in Figure 1, A6237? The detected bands are a little lower than on several other immunoblotting images.
It would be useful to discuss the extra bands (ARP40981, NBP3-13791) and the unexpected band sizes (NBP2-53336) in immunoblotting.
In “Methods”, “Cell culture”: I believe that “2 mM L-glutamate (Wisent cat. number 609065)” is L-glutamine.
“Methods”, “Antibody screening by Western Blot”: “... protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429)”: I guess that you refer to the Halt™ Protease Inhibitor Cocktail, but that is a Thermo Fisher Scientific product.
You wrote that the lysates were cleared with 110,000 x g centrifugation. Could you, please, confirm that? That is an ultracentrifugation speed. Lysates are usually cleared with centrifugal forces about an order of magnitude lower for lysate preparation for immunoblotting and immunoprecipitation.
I advise against using the wavy line “~” in a method paper. I believe that it reads “about” or “approximately”. Instead, please just state the numbers.
“Methods”, “Antibody screening by immunoprecipitation”: please state the dilution of the P8340 protease inhibitor cocktail.
“Methods”, “Antibody screening by immunoprecipitation”: please describe how you eluted the proteins from the immunoprecipitation beads.
“Methods”, “Antibody screening by immunofluorescence”: the DAPI staining is mentioned early in the protocol, and then later again, when the secondary antibody staining is described (without stating the DAPI concentration). I would not mention DAPI before the plating step because it could confuse the readers that it was used alongside the CellTracker stains.
“DAPI (Thermo Fisher Scientific, cat. Number D3571)”: that, however, is a Sigma-Aldrich product.
None of the TIA1 KO cells were completely dark in the immunofluorescence studies in Figure 3. It would be a good idea to point out that more stringent immunofluorescence conditions may be needed.

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?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biochemistry, cell biology, neurodegeneration

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 to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. We ... Continue reading
Thank you to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. 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 address your general concerns, we’d first like to clarify that this study for TIA1 is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That is why we have focused our initiative on testing antibodies in human cancer cell lines rather than murine models, but are well aware that many researchers employ these models. YCharOS presents the antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies. This enables researchers who specialize in the target of interest to conduct further studies, including employing these high-performing antibodies on murine cell lines.

Furthermore, the objective of this article, as well as the YCharOS initiative as a whole, is not to interpret the results nor summarize the performance of the antibodies tested. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. WE’d like to re-iterate that 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. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, and results&discussion section that ensure our goals are aligned and defined to all readers. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies would be valid only under the precise experimental setup and cell line used.

To address your minor comments, the authors have decided to stick with using the term Western blot to remain consistent with all other characterization reports. As for your concern regarding TIAR cross-reactivity, each commercial antibody tested was advertised on their respective catalogs as targeting TIA1, rather than TIAR. From what we’ve found in literature, TIAR is encoded by a different gene, being TIAL (1-3). For us to make accurate conclusions regarding the cross reactivity of the commercial antibodies, we would have had to prepare two KO cell lines; TIA1 and TIAL.

In the newly submitted version of this article, additional references have been included to address TIA1’s role in various diseased states.

The molecular weight marker has been confirmed the authors. Unlike other antibodies tested, A6237 recognizes bands in the WT cell line that do not disappear in the KO cell lines, indicating that it might not be properly targeting TIA1. When analyzing an antibody by Western blot, if it recognizes the target protein but produces extra bands as well, it is considered non-selective but specific. If it fails to recognize the target protein and produces unexpected bands, it is considered non-specific. Guidelines on how to analyze antibody characterization data can be found in an editorial by Biddle et al., featured on the YCharOS gateway (4). In the newly submitted version of this article, we will include this reference.

The following responses address your concerns regarding the methods section:

L-glutamate has been changed to L-glutamine.

The MilliporeSigma protease inhibitor cocktail mentioned is the one used in our standardized protocol. In our efforts to be more accurate, we have removed the word “mix” in the manuscript to reflect how it is written in the catalog.

WB: We found that ultra-centrifugation speed is required to pellet insoluble contaminants found in the lysates that would adhere to the bead-antibody conjugate, interfering with the detection of bound protein in Western blot. We’ve observed that Table-top centrifugation speeds do not adequately remove insoluble particles.

IP: the concentration for the protease inhibitor cocktail mix as well as a statement describing how the protein of interest can be eluded using the antibody-bead conjugation protocol has been added.

IF: we’ve relocated the mention of DAPI fluorescent stain to where secondary antibody conjugation is discussed to prevent confusion among readers. In this case, the DAPI stain was purchased from Thermo Fisher Scientific, which is why it is mentioned in the manuscript.

Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629-39. doi: 10.1016/0092-8674(91)90536-8.

Velasco BR, Izquierdo JM. T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology. Int J Mol Sci. 2022 Jul 16;23(14):7836. doi: 10.3390/ijms23147836.

Beck AR, Medley QG, O'Brien S, Anderson P, Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829-35. doi: 10.1093/nar/24.19.3829.

Biddle MS and Virk HS. YCharOS open antibody characterisation data: Lessons learned and progress made [version 1; peer review: not peer reviewed]. F1000Research 2023, 12:1344 (https://doi.org/10.12688/f1000research.141719.1)
Thank you to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. 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 address your general concerns, we’d first like to clarify that this study for TIA1 is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That is why we have focused our initiative on testing antibodies in human cancer cell lines rather than murine models, but are well aware that many researchers employ these models. YCharOS presents the antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies. This enables researchers who specialize in the target of interest to conduct further studies, including employing these high-performing antibodies on murine cell lines.

Furthermore, the objective of this article, as well as the YCharOS initiative as a whole, is not to interpret the results nor summarize the performance of the antibodies tested. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. WE’d like to re-iterate that 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. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, and results&discussion section that ensure our goals are aligned and defined to all readers. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies would be valid only under the precise experimental setup and cell line used.

To address your minor comments, the authors have decided to stick with using the term Western blot to remain consistent with all other characterization reports. As for your concern regarding TIAR cross-reactivity, each commercial antibody tested was advertised on their respective catalogs as targeting TIA1, rather than TIAR. From what we’ve found in literature, TIAR is encoded by a different gene, being TIAL (1-3). For us to make accurate conclusions regarding the cross reactivity of the commercial antibodies, we would have had to prepare two KO cell lines; TIA1 and TIAL.

In the newly submitted version of this article, additional references have been included to address TIA1’s role in various diseased states.

The molecular weight marker has been confirmed the authors. Unlike other antibodies tested, A6237 recognizes bands in the WT cell line that do not disappear in the KO cell lines, indicating that it might not be properly targeting TIA1. When analyzing an antibody by Western blot, if it recognizes the target protein but produces extra bands as well, it is considered non-selective but specific. If it fails to recognize the target protein and produces unexpected bands, it is considered non-specific. Guidelines on how to analyze antibody characterization data can be found in an editorial by Biddle et al., featured on the YCharOS gateway (4). In the newly submitted version of this article, we will include this reference.

The following responses address your concerns regarding the methods section:

L-glutamate has been changed to L-glutamine.

The MilliporeSigma protease inhibitor cocktail mentioned is the one used in our standardized protocol. In our efforts to be more accurate, we have removed the word “mix” in the manuscript to reflect how it is written in the catalog.

WB: We found that ultra-centrifugation speed is required to pellet insoluble contaminants found in the lysates that would adhere to the bead-antibody conjugate, interfering with the detection of bound protein in Western blot. We’ve observed that Table-top centrifugation speeds do not adequately remove insoluble particles.

IP: the concentration for the protease inhibitor cocktail mix as well as a statement describing how the protein of interest can be eluded using the antibody-bead conjugation protocol has been added.

IF: we’ve relocated the mention of DAPI fluorescent stain to where secondary antibody conjugation is discussed to prevent confusion among readers. In this case, the DAPI stain was purchased from Thermo Fisher Scientific, which is why it is mentioned in the manuscript.

Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629-39. doi: 10.1016/0092-8674(91)90536-8.

Velasco BR, Izquierdo JM. T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology. Int J Mol Sci. 2022 Jul 16;23(14):7836. doi: 10.3390/ijms23147836.

Beck AR, Medley QG, O'Brien S, Anderson P, Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829-35. doi: 10.1093/nar/24.19.3829.

Biddle MS and Virk HS. YCharOS open antibody characterisation data: Lessons learned and progress made [version 1; peer review: not peer reviewed]. F1000Research 2023, 12:1344 (https://doi.org/10.12688/f1000research.141719.1)
Competing Interests: No competing interests were disclosed. Close
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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 to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. We ... Continue reading
Thank you to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. 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 address your general concerns, we’d first like to clarify that this study for TIA1 is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That is why we have focused our initiative on testing antibodies in human cancer cell lines rather than murine models, but are well aware that many researchers employ these models. YCharOS presents the antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies. This enables researchers who specialize in the target of interest to conduct further studies, including employing these high-performing antibodies on murine cell lines.

Furthermore, the objective of this article, as well as the YCharOS initiative as a whole, is not to interpret the results nor summarize the performance of the antibodies tested. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. WE’d like to re-iterate that 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. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, and results&discussion section that ensure our goals are aligned and defined to all readers. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies would be valid only under the precise experimental setup and cell line used.

To address your minor comments, the authors have decided to stick with using the term Western blot to remain consistent with all other characterization reports. As for your concern regarding TIAR cross-reactivity, each commercial antibody tested was advertised on their respective catalogs as targeting TIA1, rather than TIAR. From what we’ve found in literature, TIAR is encoded by a different gene, being TIAL (1-3). For us to make accurate conclusions regarding the cross reactivity of the commercial antibodies, we would have had to prepare two KO cell lines; TIA1 and TIAL.

In the newly submitted version of this article, additional references have been included to address TIA1’s role in various diseased states.

The molecular weight marker has been confirmed the authors. Unlike other antibodies tested, A6237 recognizes bands in the WT cell line that do not disappear in the KO cell lines, indicating that it might not be properly targeting TIA1. When analyzing an antibody by Western blot, if it recognizes the target protein but produces extra bands as well, it is considered non-selective but specific. If it fails to recognize the target protein and produces unexpected bands, it is considered non-specific. Guidelines on how to analyze antibody characterization data can be found in an editorial by Biddle et al., featured on the YCharOS gateway (4). In the newly submitted version of this article, we will include this reference.

The following responses address your concerns regarding the methods section:

L-glutamate has been changed to L-glutamine.

The MilliporeSigma protease inhibitor cocktail mentioned is the one used in our standardized protocol. In our efforts to be more accurate, we have removed the word “mix” in the manuscript to reflect how it is written in the catalog.

WB: We found that ultra-centrifugation speed is required to pellet insoluble contaminants found in the lysates that would adhere to the bead-antibody conjugate, interfering with the detection of bound protein in Western blot. We’ve observed that Table-top centrifugation speeds do not adequately remove insoluble particles.

IP: the concentration for the protease inhibitor cocktail mix as well as a statement describing how the protein of interest can be eluded using the antibody-bead conjugation protocol has been added.

IF: we’ve relocated the mention of DAPI fluorescent stain to where secondary antibody conjugation is discussed to prevent confusion among readers. In this case, the DAPI stain was purchased from Thermo Fisher Scientific, which is why it is mentioned in the manuscript.

Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629-39. doi: 10.1016/0092-8674(91)90536-8.

Velasco BR, Izquierdo JM. T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology. Int J Mol Sci. 2022 Jul 16;23(14):7836. doi: 10.3390/ijms23147836.

Beck AR, Medley QG, O'Brien S, Anderson P, Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829-35. doi: 10.1093/nar/24.19.3829.

Biddle MS and Virk HS. YCharOS open antibody characterisation data: Lessons learned and progress made [version 1; peer review: not peer reviewed]. F1000Research 2023, 12:1344 (https://doi.org/10.12688/f1000research.141719.1)
Thank you to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. 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 address your general concerns, we’d first like to clarify that this study for TIA1 is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That is why we have focused our initiative on testing antibodies in human cancer cell lines rather than murine models, but are well aware that many researchers employ these models. YCharOS presents the antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies. This enables researchers who specialize in the target of interest to conduct further studies, including employing these high-performing antibodies on murine cell lines.

Furthermore, the objective of this article, as well as the YCharOS initiative as a whole, is not to interpret the results nor summarize the performance of the antibodies tested. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. WE’d like to re-iterate that 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. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, and results&discussion section that ensure our goals are aligned and defined to all readers. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies would be valid only under the precise experimental setup and cell line used.

To address your minor comments, the authors have decided to stick with using the term Western blot to remain consistent with all other characterization reports. As for your concern regarding TIAR cross-reactivity, each commercial antibody tested was advertised on their respective catalogs as targeting TIA1, rather than TIAR. From what we’ve found in literature, TIAR is encoded by a different gene, being TIAL (1-3). For us to make accurate conclusions regarding the cross reactivity of the commercial antibodies, we would have had to prepare two KO cell lines; TIA1 and TIAL.

In the newly submitted version of this article, additional references have been included to address TIA1’s role in various diseased states.

The molecular weight marker has been confirmed the authors. Unlike other antibodies tested, A6237 recognizes bands in the WT cell line that do not disappear in the KO cell lines, indicating that it might not be properly targeting TIA1. When analyzing an antibody by Western blot, if it recognizes the target protein but produces extra bands as well, it is considered non-selective but specific. If it fails to recognize the target protein and produces unexpected bands, it is considered non-specific. Guidelines on how to analyze antibody characterization data can be found in an editorial by Biddle et al., featured on the YCharOS gateway (4). In the newly submitted version of this article, we will include this reference.

The following responses address your concerns regarding the methods section:

L-glutamate has been changed to L-glutamine.

The MilliporeSigma protease inhibitor cocktail mentioned is the one used in our standardized protocol. In our efforts to be more accurate, we have removed the word “mix” in the manuscript to reflect how it is written in the catalog.

WB: We found that ultra-centrifugation speed is required to pellet insoluble contaminants found in the lysates that would adhere to the bead-antibody conjugate, interfering with the detection of bound protein in Western blot. We’ve observed that Table-top centrifugation speeds do not adequately remove insoluble particles.

IP: the concentration for the protease inhibitor cocktail mix as well as a statement describing how the protein of interest can be eluded using the antibody-bead conjugation protocol has been added.

IF: we’ve relocated the mention of DAPI fluorescent stain to where secondary antibody conjugation is discussed to prevent confusion among readers. In this case, the DAPI stain was purchased from Thermo Fisher Scientific, which is why it is mentioned in the manuscript.

Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629-39. doi: 10.1016/0092-8674(91)90536-8.

Velasco BR, Izquierdo JM. T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology. Int J Mol Sci. 2022 Jul 16;23(14):7836. doi: 10.3390/ijms23147836.

Beck AR, Medley QG, O'Brien S, Anderson P, Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829-35. doi: 10.1093/nar/24.19.3829.

Biddle MS and Virk HS. YCharOS open antibody characterisation data: Lessons learned and progress made [version 1; peer review: not peer reviewed]. F1000Research 2023, 12:1344 (https://doi.org/10.12688/f1000research.141719.1)
Competing Interests: No competing interests were disclosed. Close
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Reviewer Report 07 Sep 2023

Claudia Fallini, George and Anne Ryan Institute for Neuroscience, University of Rhode Island, Kingston, Rhode Island, USA

Approved

https://doi.org/10.5256/f1000research.146653.r195326

In this manuscript, Fotouhi and colleagues characterized a panel of 12 antibodies for their ability to detect the RNA-binding protein TIA-1 in western blot, immunoprecipitation, and immunofluorescence assays. They selected the human cancer line HAP1 as model system as it produces high levels of the TIA-1 protein, and compared data to the isogenic TIA-1 KO line. The experimental procedures are well described and thorough, and the data are solidly presented. A couple of points that the authors could address are listed below:

1. All experiments are performed on a human cell line. The authors should specify this limitation in their results and discussion section. Alternatively the authors could test the best performing antibodies on murine cells as well, as this would broaden the applicability of the study.

2. A summary table ranking the antibodies tested based on their performance on the three different assays would be helpful.

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?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Cell biology, neurodegenerative research

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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Claudia Fallini, University of Rhode Island, Kingston, USA
Jozsef Gal, University of Kentucky, Lexington, USA; University of Kentucky, Lexington, USA

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17 Apr 2024 | for Version 2

Jozsef Gal, Radiation Medicine, University of Kentucky, Lexington, Kentucky, USA; Markey Cancer Center, University of Kentucky, Lexington, Kentucky, USA

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Approved

I have no further comments to make.

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No competing interests were disclosed.

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Biochemistry, cell biology, neurodegeneration

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20 Mar 2024 | for Version 1

Jozsef Gal, Radiation Medicine, University of Kentucky, Lexington, Kentucky, USA; Markey Cancer Center, University of Kentucky, Lexington, Kentucky, USA

15 Views Cite this report Responses(1)

Approved With Reservations

The HAP1 cell line is of human origin. A lot of research employs murine models and cell lines. It would be very useful if the Authors could perform immunoblotting of a lysate from a murine cell line. I would recommend N2A/Neuro-2A cells. In my opinion, the antibodies that performed the best in HAP1 immunoblotting (ab140595, ab263945, NBP2-67203, 86050) would be sufficient.
The Authors present their findings in a rather raw format without much interpretation. It would be great if the Authors would summarize their findings after each technique. It would also be very useful to provide a summary paragraph about which antibodies the Authors consider overall the best.

Minor comments:

The term “Western blot” is kind of colloquial. I recommend the term “immunoblotting”, but I would like to leave it up to the Authors.
TIA-1 has a close homolog in human cells, TIAR. To me, one of the most valuable contributions of this work is the ability to see which tested antibodies may cross-react with TIAR. Based on the immunoblotting image, A6237, GTX33545, and MA5-26474 may cross-react with TIAR. I think that it would be useful if the Authors would touch on this issue.
In the Introduction: “... disrupting the function of TIA1 can lead to various diseases including cancer, autoimmune diseases and neurodegenerative disorders” – I recommend including further references in this paragraph for cancer and autoimmune diseases.
Could you, please, check the molecular weight marker bands in Figure 1, A6237? The detected bands are a little lower than on several other immunoblotting images.
It would be useful to discuss the extra bands (ARP40981, NBP3-13791) and the unexpected band sizes (NBP2-53336) in immunoblotting.
In “Methods”, “Cell culture”: I believe that “2 mM L-glutamate (Wisent cat. number 609065)” is L-glutamine.
“Methods”, “Antibody screening by Western Blot”: “... protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429)”: I guess that you refer to the Halt™ Protease Inhibitor Cocktail, but that is a Thermo Fisher Scientific product.
You wrote that the lysates were cleared with 110,000 x g centrifugation. Could you, please, confirm that? That is an ultracentrifugation speed. Lysates are usually cleared with centrifugal forces about an order of magnitude lower for lysate preparation for immunoblotting and immunoprecipitation.
I advise against using the wavy line “~” in a method paper. I believe that it reads “about” or “approximately”. Instead, please just state the numbers.
“Methods”, “Antibody screening by immunoprecipitation”: please state the dilution of the P8340 protease inhibitor cocktail.
“Methods”, “Antibody screening by immunoprecipitation”: please describe how you eluted the proteins from the immunoprecipitation beads.
“Methods”, “Antibody screening by immunofluorescence”: the DAPI staining is mentioned early in the protocol, and then later again, when the secondary antibody staining is described (without stating the DAPI concentration). I would not mention DAPI before the plating step because it could confuse the readers that it was used alongside the CellTracker stains.
“DAPI (Thermo Fisher Scientific, cat. Number D3571)”: that, however, is a Sigma-Aldrich product.
None of the TIA1 KO cells were completely dark in the immunofluorescence studies in Figure 3. It would be a good idea to point out that more stringent immunofluorescence conditions may be needed.

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?

Partly
Are the datasets clearly presented in a useable and accessible format?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biochemistry, cell biology, neurodegeneration

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Responses (1)

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 to Jozsef Gal for your extensive review of this article. We have submitted a modified version of this manuscript, with the requested modifications the authors found appropriate. 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 address your general concerns, we’d first like to clarify that this study for TIA1 is part of a much larger collaborative initiative, seeking to characterize antibodies for all human proteins to address the antibody liability crisis. It would be very difficult to optimize every working parameter when in the process of trying to characterize antibodies for 20,000 proteins in the human proteome. That is why we have focused our initiative on testing antibodies in human cancer cell lines rather than murine models, but are well aware that many researchers employ these models. YCharOS presents the antibody characterization data to the scientific community, using a standardized protocol that allows researchers to select high-performing antibodies. This enables researchers who specialize in the target of interest to conduct further studies, including employing these high-performing antibodies on murine cell lines.

Furthermore, the objective of this article, as well as the YCharOS initiative as a whole, is not to interpret the results nor summarize the performance of the antibodies tested. Given that it is formatted as a Data Note, it does not require the results to be discussed or concluded. WE’d like to re-iterate that 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. That being said, we understand how this intention may be misinterpreted. A new version has been submitted, to include modifications to the title, and results&discussion section that ensure our goals are aligned and defined to all readers. Moreover, as the antibodies are tested under one specific set of conditions, summarizing the performance of the antibodies would be valid only under the precise experimental setup and cell line used.

To address your minor comments, the authors have decided to stick with using the term Western blot to remain consistent with all other characterization reports. As for your concern regarding TIAR cross-reactivity, each commercial antibody tested was advertised on their respective catalogs as targeting TIA1, rather than TIAR. From what we’ve found in literature, TIAR is encoded by a different gene, being TIAL (1-3). For us to make accurate conclusions regarding the cross reactivity of the commercial antibodies, we would have had to prepare two KO cell lines; TIA1 and TIAL.

In the newly submitted version of this article, additional references have been included to address TIA1’s role in various diseased states.

The molecular weight marker has been confirmed the authors. Unlike other antibodies tested, A6237 recognizes bands in the WT cell line that do not disappear in the KO cell lines, indicating that it might not be properly targeting TIA1. When analyzing an antibody by Western blot, if it recognizes the target protein but produces extra bands as well, it is considered non-selective but specific. If it fails to recognize the target protein and produces unexpected bands, it is considered non-specific. Guidelines on how to analyze antibody characterization data can be found in an editorial by Biddle et al., featured on the YCharOS gateway (4). In the newly submitted version of this article, we will include this reference.

The following responses address your concerns regarding the methods section:

L-glutamate has been changed to L-glutamine.
The MilliporeSigma protease inhibitor cocktail mentioned is the one used in our standardized protocol. In our efforts to be more accurate, we have removed the word “mix” in the manuscript to reflect how it is written in the catalog.
WB: We found that ultra-centrifugation speed is required to pellet insoluble contaminants found in the lysates that would adhere to the bead-antibody conjugate, interfering with the detection of bound protein in Western blot. We’ve observed that Table-top centrifugation speeds do not adequately remove insoluble particles.
IP: the concentration for the protease inhibitor cocktail mix as well as a statement describing how the protein of interest can be eluded using the antibody-bead conjugation protocol has been added.
IF: we’ve relocated the mention of DAPI fluorescent stain to where secondary antibody conjugation is discussed to prevent confusion among readers. In this case, the DAPI stain was purchased from Thermo Fisher Scientific, which is why it is mentioned in the manuscript.

Tian Q, Streuli M, Saito H, Schlossman SF, Anderson P. A polyadenylate binding protein localized to the granules of cytolytic lymphocytes induces DNA fragmentation in target cells. Cell. 1991 Nov 1;67(3):629-39. doi: 10.1016/0092-8674(91)90536-8.
Velasco BR, Izquierdo JM. T-Cell Intracellular Antigen 1-Like Protein in Physiology and Pathology. Int J Mol Sci. 2022 Jul 16;23(14):7836. doi: 10.3390/ijms23147836.
Beck AR, Medley QG, O'Brien S, Anderson P, Streuli M. Structure, tissue distribution and genomic organization of the murine RRM-type RNA binding proteins TIA-1 and TIAR. Nucleic Acids Res. 1996 Oct 1;24(19):3829-35. doi: 10.1093/nar/24.19.3829.
Biddle MS and Virk HS. YCharOS open antibody characterisation data: Lessons learned and progress made [version 1; peer review: not peer reviewed]. F1000Research 2023, 12:1344 (https://doi.org/10.12688/f1000research.141719.1)

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Competing Interests

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Reviewer Report

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07 Sep 2023 | for Version 1

Claudia Fallini, George and Anne Ryan Institute for Neuroscience, University of Rhode Island, Kingston, Rhode Island, USA

10 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?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cell biology, neurodegenerative research

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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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 RNA-binding protein TIA1 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. TIA1 antibody screening by Western Blot.

Figure 2. TIA1 antibody screening by immunoprecipitation.

Figure 3. TIA1 antibody screening by immunofluorescence.

Methods

Antibodies

Table 2. Summary of the TIA1 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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