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

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.


Introduction
2][3][4] Suppressing translation in environmentally stressed cells and promoting stress granule formation, TIA1 modulates cellular response to stress and inflammation. 5,62][13][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. 6,11Significant efforts are required to further elucidate the relationship between dysregulated RNA metabolism and ALS/FTD pathogenesis which may lead to novel therapeutic discoveries. 11,15chanistic 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
7][18][19][20][21][22][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 log 2 (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).
[19][20][21][22][23] For immunoprecipitation experiments, we used the antibodies to immunopurify TIA1 from HAP1 cell extracts.[19][20][21][22][23] For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 24In 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).
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

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.
Any further responses from the reviewers can be found at the end of the article 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. 25The underlying data can be found on Zenodo open access repository. 26,27

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. 28Peroxidase-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 antimouse secondary antibodies are from Thermo Fisher Scientific (cat.number A21429 and A21424).

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   Antibody screening by Western Blot Western Blots were performed as described in our standard operating procedure. 30HAP1 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. 31Antibody-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.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 manuscript is well written.This is a very useful contribution to the field.It will certainly help researchers choose anti-TIA1 antibodies for future work.I only have minor issues and comments.

General issues:
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.

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The Authors present their findings in a rather raw format without much interpretation.It would 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.

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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 crossreact with TIAR.I think that it would be useful if the Authors would touch on this issue.

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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.

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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.

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It would be useful to discuss the extra bands (ARP40981, NBP3-13791) and the unexpected band sizes (NBP2-53336) in immunoblotting.
○ "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.

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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, 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.
○ 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.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.
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 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.1.
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 it is written in the catalog.

2.
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.

3.
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.

4.
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.

5.
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.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 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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Figure 2 .
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.

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Is the rationale for creating the dataset(s) clearly described?YesAre the protocols appropriate and is the work technically sound?PartlyAre sufficient details of methods and materials provided to allow replication by others?PartlyAre the datasets clearly presented in a useable and accessible format?PartlyCompeting Interests: No competing interests were disclosed.

Table 1 .
Summary of the cell lines used.
Â 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 Â 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/0.45NA 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.