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Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 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.

Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson’s disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 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.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS 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.

SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer’s disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-1 commercial antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

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.

Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue 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.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is 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 the 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.

Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue 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.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 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.

Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer’s disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK 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.

Background A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.

The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer’s Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Secreted frizzled-related protein 1 (sFRP-1) is a secreted protein, belonging to the secreted glycoprotein SFRP family. As a modulator of the Wnt/β-catenin signalling pathway, sFRP-1 has implications in human cancers and neurological diseases. If the community had access to well-characterized anti-sFRP-1 antibodies, the reproducibility of sFRP-1 research would be enhanced. In this study, we characterized 11 sFRP-1 commercial antibodies for Western Blot and immunoprecipitation, 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 the antibody reproducibility issue 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.

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.

Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in 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.

Rab1 is a highly conserved small GTPase that exists in humans as two isoforms: Rab1A and Rab1B, sharing 92% sequence identity. These proteins regulate vesicle trafficking between the endoplasmic reticulum (ER) and Golgi and within the Golgi stacks. Rab1A and Rab1B may be oncogenes, as they are frequently dysregulated in various human cancers. Moreover, they contribute to the progression of Parkinson’s disease. The availability of high-quality antibodies specific for Rab1A or Rab1B is essential to understand the distinct functions of these Rab1 proteins in both health and diseaseand to enhance the reproducibility of research involving these proteins. In this study, we characterized seven antibodies targeting Rab1A and five antibodies targeting Rab1B 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 much larger, collaborative initiative seeking to address the antibody reproducibility issue by characterizing commercially available antibodies for human proteins and publishing the results openly as a valuable resource for the scientific community. While uses 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.

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we have characterized ten Moesin commercial antibodies in 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 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.

Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (ii) yet, ~50–75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and (iii) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts.

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson’s Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 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.

Apolipoprotein E is a secreted protein involved in mediating lipid distribution and metabolism among cells of specific tissues. The dysregulation of Apolipoprotein E can disturb cholesterol homeostasis, resulting in several diseases, including cardiovascular disease and Alzheimer’s disease. The therapeutic potential of Apolipoprotein E against these diseases demonstrates the importance of providing high-quality antibodies for this protein to the scientific community. In this study, we characterized fourteen Apolipoprotein E commercial antibodies for Western Blot and immunoprecipitation, 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.

Superoxide dismutase [Cu-Zn] 1 (SOD1), is an antioxidant enzyme encoded by the gene SOD1, responsible for regulating oxidative stress levels by sequestering free radicals. Identified as the first gene with mutations in Amyotrophic lateral sclerosis (ALS), SOD1 is a determinant for studying diseases of aging and neurodegeneration. With guidance on well-characterized anti-SOD1 antibodies, the reproducibility of SOD1 research would be enhanced. In this study, we characterized eleven SOD1 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.

CHCHD10 is a mitochondrial protein, implicated in the regulation of mitochondrial morphology and cristae structure, as well as the maintenance of mitochondrial DNA integrity. Recently discovered to be associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in its mutant form, the scientific community would benefit from the availability of validated anti-CHCHD10 antibodies. In this study, we characterized four CHCHD10 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. As this study highlights high-performing antibodies for CHCHD10, we encourage readers to use it as a guide to select the most appropriate antibody for their specific needs.

Charged multivesicular body protein 2B is a subunit of the endosomal sorting complex required for transport III (ESRCT-III), a complex implicated in the lysosomal degradation pathway and formation of multivesicular bodies. Mutations to the CHMP2B gene can result in abnormal protein aggregates in neurons and is therefore predicted to be associated in neurodegenerative diseases, including across the ALS-FTD spectrum. Through our standardized experimental protocol which compares read-outs in knockout cell lines and isogenic parental controls, this study aims to enhance the reproducibility of research on this target by characterizing eight commercial antibodies against charged multivesicular body protein 2b using Western Blot, immunoprecipitation, and immunofluorescence. 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.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS 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.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein playing a critical role in the regulation of transcription, splicing and RNA stability. Mutations in TARDBP leading to aggregation, are suspected to be a characteristic feature of various neurogenerative diseases. The lack of well-characterized anti- TDP-43 antibodies acts as a barrier to establish reproducible TDP-43 research. In this study, we characterized eighteen TDP-43 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 well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Profilin-1, a member of the Profilin family, is a ubiquitously expressed protein that controls actin polymerization in a concentration-dependent manner. As mutations in the Profilin-1 gene have potential implications in neurodegenerative disease progression, well-characterized anti-Profilin-1 antibodies would be beneficial to the scientific community. In this study, we characterized sixteen Profilin-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence applications, 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.

Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance in 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.

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein playing a critical role in the regulation of transcription, splicing and RNA stability. Mutations in TARDBP leading to aggregation, are suspected to be a characteristic feature of various neurogenerative diseases. The lack of well-characterized anti- TDP-43 antibodies acts as a barrier to establish reproducible TDP-43 research. In this study, we characterized eighteen TDP-43 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 well-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 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.

TBK1 is a serine-threonine protein kinase that has been linked to a number of diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for TBK1 for use in immunoblot, immunofluorescence and immunoprecipitation, using an isogeneic knock-out cell line as a control. We identify antibodies that appear specific for all three applications but invite the readers to interpret the present findings based on their own scientific expertise and use this report as a guide to select the most appropriate antibody for their specific needs.

TBK1 is a serine-threonine protein kinase that has been linked to a number of diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Reproducible research on TBK1 has been hampered by the lack of well characterized antibodies. In this study, we characterized 11 commercial antibodies for TBK1 for use in immunoblot, immunofluorescence and immunoprecipitation, using an isogeneic knock-out cell line as a control. We identify antibodies that appear specific for all three applications but invite the readers to interpret the present findings based on their own scientific expertise and use this report as a guide to select the most appropriate antibody for their specific needs.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS 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.

Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson’s disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 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.

STING1 is an immune adaptor protein which promotes innate immune defense mechanisms against pathogens. Its role in modulating inflammation links STING1 to various pathologic conditions, positioning it as a key target for therapeutic interventions aimed at regulating immune responses. To advance our understanding of STING1-associated diseases, it is essential to make high-performing antibodies readily accessible to the scientific community. This study aims to improve reliability of STING1 research as we have characterized sixteen STING1 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.

SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer’s disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-1 commercial antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-coupled protein receptor that induces crucial biological processes when bound by sphingosine 1-phosphate. Here, we have characterized nine S1PR1 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.

Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 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.

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.

Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue 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.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is 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 the 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.

Midkine is a secreted protein that acts as a growth factor or cytokine involved in cell survival and inflammatory processes. It accumulates in amyloid plaques, which are hallmarks of Alzheimer’s Disease (AD). The reproducibility of Midkine research would be enhanced if the community had access to well-characterized anti-Midkine antibodies. In this study, we characterized 8 commercial Midkine antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in a knockout cell line and isogenic parental control. These studies are part of a larger, collaborative initiative seeking to address the antibody reproducibility issue 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.

Synaptotagmin-1 is a synaptic vesicle transmembrane protein that senses calcium influx via its tandem C2-domains, triggering synchronous neurotransmitter release. Disruption to SYT1 is associated with neurodevelopmental disorders, highlighting the importance of identifying high-quality research reagents to enhance understanding of Synaptotagmin-1 in health and disease. Here we have characterized thirteen Synaptotagmin-1 commercial antibodies for western blot, immunoprecipitation, immunofluorescence and flow cytometry using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G-coupled protein receptor that induces crucial biological processes when bound by sphingosine 1-phosphate. Here, we have characterized nine S1PR1 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.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 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.

Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 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.

Protein phosphatase 2A is a serine/threonine phosphatase with activity dependent on an associated regulatory subunit, serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D). PPP2R5D is the δ isoform in the B56 family of regulatory subunits. Abundantly expressed in the brain and involved in a broad range of cellular processes, PPP2R5D plays an essential role in modulating key neuronal pathways and signalling. Pathogenic mutations in the PPP2R5D gene are linked to clinical symptoms characterized by neurodevelopmental delay, intellectual disability, and autism spectrum disorders. The etiology of these genetic disorders remains unknown, which can partly be due to the lack of independently characterized antibodies. Here we have characterized six PPP2R5D 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 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.

Tyrosine-protein kinase SYK, encoded by the SYK gene, is a non-receptor type protein kinase which mediates immune signal transduction through immunoreceptors. Tyrosine-protein kinase SYK expression has been associated with the development of various inflammatory diseases, cancer and neurodegenerative conditions. The reproducibility of tyrosine-protein kinase SYK research would help elucidate the mechanism in which it causes neuroinflammation as well as its potential as a novel target to treat Alzheimer’s disease. This would be facilitated with the availability of high-quality tyrosine-protein kinase SYK. In this study, we characterized thirteen tyrosine-protein kinase SYK 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.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is 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 the 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.

Background A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.