
==== Front
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F1000Res
F1000Research
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10.12688/f1000research.153670.1
Data Note
Articles
A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence
[version 1; peer review: 2 approved]

Fanti Rebeka Investigation Validation Writing – Original Draft Preparation 1
Ayoubi Riham Data Curation Investigation Methodology Validation Visualization Writing – Original Draft Preparation 2
Alende Charles Investigation Visualization https://orcid.org/0009-0005-4611-6134
2
Fotouhi Maryam Investigation 2
González Bolívar Sara Investigation https://orcid.org/0000-0002-4299-8281
2
Chandrasekaran Renu Investigation 1
Southern Kathleen Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4125-3608
2
Edwards Aled M. Funding Acquisition Supervision 1
Harding Rachel J. Supervision Validation https://orcid.org/0000-0002-1134-391X
13
Laflamme Carl Funding Acquisition Methodology Project Administration Resources Supervision https://orcid.org/0000-0001-5906-025X
a2
NeuroSGC/YCharOS/EDDU collaborative group
ABIF consortium
1 Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada
2 Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, Canada
3 Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada
a carl.laflamme@mcgill.ca
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.

13 8 2024
2024
13 92216 7 2024
Copyright: © 2024 Fanti R et al.
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

UniProt ID P42858
HTT
Huntingtin
antibody characterization
antibody validation
western blot
immunoprecipitation
immunofluorescence
Genome CanadaMitacsOntario GenomicsOGI-210 Genome QuebecThis work was supported by a grant from the Government of Canada through Genome Canada, Genome Quebec, and Ontario Genomics (grant no. OGI-210). RF and RA are supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
==== Body
pmcIntroduction

Huntington’s Disease (HD) is a neurodegenerative disorder inherited in an autosomal dominant manner, presenting with a spectrum of progressive motor, cognitive, and psychological impairments, typically with adult-onset of symptoms. 1 Although the HD causative gene, HTT, was discovered over three decades ago, there are still no disease-modifying treatments available for patients, and progress unpicking the molecular pathology of the disease remains slow. 2

HD arises from a heterozygous expansion mutation of the trinucleotide CAG repeat tract in exon 1 of HTT, located on chromosome 4, above a critical threshold of ~36 repeats. This mutation results in expansion of the polyglutamine stretch at the N-terminus of the 3144 amino acid Huntingtin protein. Huntingtin functions as a scaffold protein, engaging in extensive protein-protein interactions, 3 forming various multi-protein complexes to carry-out its diverse array of functions. Modulation of this interaction network by the polyglutamine expansion contributes to degeneration within the central nervous system, affecting medium spiny neurons at the onset of disease. 4 , 5

The low expression level, complex interactome and large size of the 348 kDa Huntingtin protein have given rise to technical challenges which have hindered precise determination of its molecular function, or how this is altered in disease. In particular, the use of different Huntingtin antibodies by scientists in the HD research community, often mapping to structurally distant epitopes, can yield different or even conflicting results, further conflating interrogation of this protein. 6 – 8

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data. 9 – 11 Here we evaluated the performance of twenty commercial antibodies for Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of Huntingtin properties and function. The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and academic representatives. It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding target protein. The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 12

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. 13

Results and discussion

Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 14 , 15 The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). We generated an HTT KO line in DMS 53 as it expresses the endogenous HTT transcript at 6.1 log 2 (TPM+1), which is above the average range of cancer cell lines analyzed. A commercial HAP1 HTT KO is also available; HAP1 expresses HTT at 3.7 log 2 (TPM+1) RNA level. A HEK293T HTT KO cell line has been developed and used elsewhere 16 ( Table 1). All three cell line backgrounds were evaluated by western blot using a high-performing Huntingtin antibody detected in Figure 1. DMS 53 was identified as the most suitable cell line ( Figure 2), which can be explained by its high expression of the HTT transcript compared to other two cell lines. Thus, DMS 53 WT and KO cell lines were generated and used to evaluate the antibodies in all applications.

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype	
ATCC	CRL-2062	CVCL_1177	DMS 53	WT	
Academic	non-commercial	CVCL_D6U0	DMS 53	HTT KO	
ATCC	CRL-3216	CVCL_0063	HEK 293T	WT	
Academic	non-commercial	CVCL_D7EP	HEK 293T	HTT KO 16	
Horizon Discovery	C631	CVCL_Y019	HAP1	WT	
Horizon Discovery	HZGHC004595c006	CVCL_SR86	HAP1	HTT KO	

Figure 1. Huntingtin antibody screening by western blot.

Lysates of DMS 53 (WT and HTT KO) were prepared and 30 μg of protein were processed for western blot with the indicated Huntingtin antibodies. Tris-Glycine 4-20% gels were used for SDS-PAGE. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies ab109115**, ab45169**, and MA5-41256** which were titrated as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab109115** at 1/2000, ab45169** at 1/5000, ABCD_AG854** at 1/10, ABCD_AG855** at 1/10, A19064** at 1/1000, 5656** at 1/1000, CH03023* at 1/1000, MW1-S* at 1/10, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/10, MW6-S* at 1/10, MW7-S* at 1/10, MW8-S* at 1/10, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/200, MA3-040* at 1/1000, MA5-16703* at 1/500, MA5-41256** at 1/2000. Predicted band size: 347 kDa. *Monoclonal antibody, **Recombinant antibody.

Note: MW1-S*, MW3-S*, MW4-S*, MW5-S*, MW6-S*, MW7-S* and MW8-S* are expected to only recognize an altered conformation of the polyQ domain generated as the polyQ domain of Huntingtin increases in length.

Figure 2. Huntingtin western blot on various cell lysates.

Lysates of WT and HTT KO in DMS 53, HEK 293T and HAP1 were prepared, and 30 μg of protein was processed for western blot with the indicated Huntingtin antibodies ab45169** at 1/5000 and GTX638832** at 1/500. Tris-Glycine 4-20% gels were used for SDS-PAGE. The Ponceau stained transfer is shown as a loading control. Predicted band size: 347 kDa. **Recombinant antibody.

For western blot experiments, WT and HTT KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with twenty Huntingtin antibodies in parallel ( Table 2, Figure 1).

Table 2. Summary of the Huntingtin antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μl)	Vendors recommended applications	
Abcam	ab109115 **	1003147-4	AB_10863082	recombinant-mono	EPR5526	rabbit	1.52	Wb, IF	
Abcam	ab45169 **	1022500	AB_733062	recombinant-mono	EP867Y	rabbit	1.55	Wb, IF	
ABCD Antibodies	ABCD_AG854 **	10/27/2023	AB_3076339	recombinant-mono	12.3	rabbit	0.004	n/a	
ABCD Antibodies	ABCD_AG855 **	10/27/2023	AB_3076340	recombinant-mono	C4	rabbit	0.19	n/a	
ABclonal	A19064 **	4000000431	AB_2862557	recombinant-mono	ARC0431	rabbit	0.63	Wb, IF	
Cell Signaling Technology	5656 **	6	AB_10827977	recombinant-mono	D7F7	rabbit	0.10	Wb, IF	
Coriell Institute	CH03023 *	03.17.21	AB_3096092	monoclonal	2B7	mouse	1.71	n/a	
DSHB	MW1-S *	44322	AB_528290	monoclonal	MW1-S	mouse	0.02	Wb, IP, IF	
DSHB	MW3-S *	43216	AB_528292	monoclonal	MW3-S	mouse	0.01	Wb, IF	
DSHB	MW4-S *	43251	AB_528293	monoclonal	MW4-S	mouse	0.02	Wb, IF	
DSHB	MW5-S *	44742	AB_528294	monoclonal	MW5-S	mouse	0.03	Wb, IF	
DSHB	MW6-S *	43230	AB_528295	monoclonal	MW6-S	mouse	0.06	Wb, IF	
DSHB	MW7-S *	43461	AB_528296	monoclonal	MW7-S	mouse	0.03	Wb, IF	
DSHB	MW8-S *	44315	AB_528297	monoclonal	MW8-S	mouse	0.04	Wb, IP, IF	
GeneTex	GTX132433	42312	AB_2886646	polyclonal	MW3-S	rabbit	1.40	Wb, IF	
GeneTex	GTX638832 **	45096	AB_3094813	recombinant-mono	HL2483	rabbit	0.98	Wb	
Thermo Fisher Scientific	710695 **	RF236710	AB_2608784	recombinant-poly	3HCLC	rabbit	0.50	IF	
Thermo Fisher Scientific	MA3-040 *	YG376237	AB_2608783	monoclonal	1HU-4C8	mouse	n/a	Wb, IF	
Thermo Fisher Scientific	MA5-16703 *	YE3913821A	AB_2538195	monoclonal	HDB4E10	mouse	1.00	Wb, IP, IF	
Thermo Fisher Scientific	MA5-41256 **	YE3913382B	AB_2899009	recombinant-mono	JB89-34	rabbit	1.00	Wb, IF	
Wb=western blot; IF=immunofluorescence; IP=immunoprecipitation; n/a=not available, DSHB=Developmental Studies Hybridoma Bank.

* Monoclonal antibody,

** Recombinant antibody.

We then assessed the capability of all twenty antibodies to capture Huntingtin from DMS 53 protein extracts using immunoprecipitation techniques, followed by western blot analysis. For the immunoblot step, a specific Huntingtin antibody identified previously (refer to Figure 1) was selected. Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE ( Figure 3).

Figure 3. Huntingtin antibody screening by immunoprecipitation.

DMS 53 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated Huntingtin antibodies pre-coupled to Dynabeads protein A or protein G. The concentration of MA3-040* is unknown and therefore 5 μL of this antibody was tested. All samples were washed and processed for western blot with the indicated Huntingtin antibody. Tris-Glycine 4-20% gels were used for SDS-PAGE. For western blot, ab45169** was used at 1/5000. 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, twenty antibodies were screened using a mosaic strategy. First, DMS 53 WT and HTT KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Huntingtin antibodies were evaluated. Both WT and KO lines were imaged in the same field of view to reduce staining, imaging and image analysis bias ( Figure 4). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, 12 and the images presented in Figure 3 are representative of this analysis.

Figure 4. Huntingtin antibody screening by immunofluorescence.

DMS 53 WT and HTT 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 on coverslips. Cells were stained with the indicated Huntingtin antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. When an antibody was recommended for immunofluorescence by the supplier, we tested it at the recommended dilution and at 1 μg/ml. The rest of the antibodies were tested at 1 and 2 μg/ml. The final concentration of each antibody was selected based on the detection range of the microscope used and a quantitative analysis not shown here. Antibody dilutions corresponding to the images shown are: ab109115** at 1/1500, ab45169** at 1/1500, ABCD_AG854** at 1/500, ABCD_AG855** at 1/200, A19064** at 1/600, 5656** at 1/100, CH03023* at 1/1700, MW1-S* at 1/20, MW3-S* at 1/10, MW4-S* at 1/10, MW5-S* at 1/15, MW6-S* at 1/60, MW7-S* at 1/15, MW8-S* at 1/20, GTX132433 at 1/500, GTX638832** at 1/500, 710695** at 1/500, MA3-040* at 1/1000, MA5-16703* at 1/1000, MA5-41256** at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened twenty commercial Huntingtin antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human DMS 53 WT and HTT KO cells. Several high-quality and renewable Huntingtin antibodies were identified in all applications. Researchers who wish to study Huntingtin in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.

The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. 17

Methods

The standardized protocols used to carry out this KO cell line-based antibody characterization platform was established and approved by a collaborative group of academics, industry researchers and antibody manufacturers. The detailed materials and step-by-step protocols used to characterize antibodies in western blot, immunoprecipitation and immunofluorescence are openly available on Protocol Exchange, a preprint server (DOI: 10.21203/rs.3.pex-2607/v1). 12

Antibodies and cell lines used

Cell lines used and primary antibodies tested in this study are listed in Tables 1 and 2, respectively. To ensure that the cell lines and antibodies are cited properly and can be easily identified, we have included their corresponding Research Resource Identifiers, or RRID. 18 , 19

Acknowledgment

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

NeuroSGC/YCharOS collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.

ABIF consortium: Claire M. Brown and Joel Ryan.

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.11582780).

Data availability

Underlying data

Zenodo: Dataset for the Huntingtin antibody screening study, doi.org/10.5281/zenodo.11639052. 17

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

10.5256/f1000research.168592.r315013
Reviewer response for version 1
Bowman Karen 1Referee
1 University of Leicester, Leicester, England, UK
9 9 2024 Copyright: © 2024 Bowman K
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Version 1recommendationapprove
The article pointed out the current technical difficulties in studying Huntington’s Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem. The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues. The study was an evaluation of twenty commercial antibodies for the protein Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence.

The study was well designed and the results were to a high technical standard and quality. The data is presented clearly and looks robust.

It was reassuring to see that a committee of industry and academic representatives had endorsed the platform used. The platform consisted of identification of human cell lines suitable for antibody characterisation studies i.e. with adequate levels of Huntingtin, followed by the development of and contribution of isogenic knockout control cell lines, which were validated at the protein level. The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies. Limitations of the study are clearly stated.

Two minor errors were spotted. There is an inconsistency between the text and figure 3, where they mention IP was done on twenty antibodies in the text, but results for only ten antibodies are presented in the figure. Perhaps some additional text on why only ten were presented would be of use, or amend the text to reflect what is presented in the figure. A second error is that in the final results paragraph on immunofluorescence, figure 3 is cited, and linked back to, instead of figure 4.

Overall, this will be a highly useful resource for scientists in the HD research community.

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

Yes

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

Yes

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

drug discovery and diagnostics

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.

10.5256/f1000research.168592.r315014
Reviewer response for version 1
Shao Jieya 1Referee https://orcid.org/0000-0003-1859-9384

1 Washington University in St Louis, St. Louis, Missouri, USA
28 8 2024 Copyright: © 2024 Shao J
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Version 1recommendationapprove
In this study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications. The experimental approaches were logically and thoroughly designed. Data are of high-quality and clearly presented. The results will be very helpful for researchers in the HTT field. However, there seems to be two small errors.

First, in Figure 3, only the IP data of 10 antibodies were shown while in the text it was stated that 20 antibodies were analyzed.

Second, on Page 5, in the last sentence of the second paragraph, the authors seem to cite Figure 3 by mistake.

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

Yes

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

Yes

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Cell biology

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.

Competing interests: No competing interests were disclosed.

Competing interests: No competing interests were disclosed.
==== Refs
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