High-quality Antibodies Research Articles

Non-endothelial expression of endomucin in the mouse and human choroid

Endomucin (EMCN) is a 261 amino acid transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn, no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn-expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn-negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in the eye.

Label-free multi-line immunochromatographic sensor based on TCBPE for broad-spectrum detection Salmonella in food

BackgroundSalmonella, a foodborne pathogen poses significant threats to food safety and human health. Immunochromatographic (ICTS) sensors have gained popularity in the field of food safety due to their convenience, speed, and cost-effectiveness. However, most existing ICTS sensors rely on antibody sandwich structures which are limited by their dependence on high-quality paired antibodies and restricted sensitivity. For the first time, we combined multi-line ICTS strips with fluorescent bacterial probes to develop a label-free multi-line immunochromatographic sensor capable of detecting broad-spectrum Salmonella. Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. ResultsUsing this sensor, we successfully detected Salmonella typhimurium within the concentration range of 104–108 CFU/mL with a visual detection limit of 6.0 × 104 CFU/mL. Compared to single-line sensors, our multi-line sensor exhibited significantly improved fluorescence intensity resulting in enhanced detection sensitivity by 50 %. Furthermore, our developed multi-line ICTS sensor demonstrated successful detection of 18 different strains of Salmonella without any cross-reaction observed with 5 common foodborne pathogens tested. The applicability and reliability were validated using milk samples, cabbage juice samples as well and drinking water samples suggesting its potential for rapid and accurate detection of Salmonella in real-world scenarios across both the food industry and clinical settings. SignificanceIn this experiment, we developed a TCBPE-based multiline immunochromatographic sensor. Specifically, Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. Through the multi-line analysis system, the detection sensitivity and accuracy of the sensor are improved. In brief, the sensor does not require complex antibody labeling and paired antibodies, and only one antibody is needed to complete the detection process.

Rapid Screening of Emergent Febuxostat Adulteration in Functional Foods Based on a Simulation-Inspired High-Quality Antibody.

Due to the potential health risks of adulterated febuxostat in uric-acid-lowering foods, it is urgent to develop rapid detection methods. However, there are no fast analytical techniques for febuxostat yet. Herein, an efficient hapten simulation strategy was proposed to successfully produce a highly sensitive and selective monoclonal antibody toward febuxostat. Based on such a robust recognition element, easy colorimetric and ultrasensitive fluorescent lateral flow immunochromatographic immunoassays were first established, which can detect febuxostat as low as 60 μg/kg by the naked eye or 1.01 μg/kg by a commercial test strip reader with acceptable stability. Furthermore, in the recovery test and blind sample analysis, consistent results between our methods and the authorized liquid chromatography-tandem mass spectrometry method suggested the high accuracy and practicality of this work. The present work not only proposes a rational hapten design idea but also provides favorable tools for the rapid screening of febuxostat in functional foods.

A Straightforward, Sensitive, and Reliable Strategy for Ethyl Carbamate Detection in By-Products from Baijiu Production by Enzyme-Linked Immunosorbent Assay.

Baijiu is a renowned Chinese distilled liquor, notable for its distinctive flavor profile and intricate production process, which prominently involves fermentation and distillation. Ethyl carbamate (EC), a probable human carcinogen, can be potentially formed during these procedures, thus prompting significant health concerns. Consequently, the contamination of EC during Baijiu production has become an increasingly pressing issue. In this study, we developed a rapid and easily operable immunoassay for determining EC in the fermented materials used in Baijiu production. The development of a high-quality antibody specific to EC facilitated a streamlined analytical procedure and heightened method sensitivity. Furthermore, we systematically evaluated other essential parameters. Following optimization, the method achieved an IC50 value of 11.83 μg/kg, with negligible cross-reactivity against EC analogs. The recovery study demonstrated the method's good accuracy and precision, with mean recovery rates ranging from 86.0% to 105.5% and coefficients of variation all below 10%. To validate the feasibility of the technique, we collected and analyzed 39 samples simultaneously using both the proposed immunoassay and confirmatory gas chromatography-mass spectrometry (GC-MS). A robust correlation was observed between the results obtained from the two methods (R2 > 0.99). The detected EC levels ranged from 2.36 μg/kg to 7.08 μg/kg, indicating an increase during the fermentation process.

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

Activates B lymphocytes and enhanced immune response: A promising adjuvant based on PLGA nanoparticle to improve the sensitivity of ZEN monoclonal antibody

In preparing monoclonal antibodies by hybridoma cell technology, the quality of B lymphocytes used for cell fusion directly affects the sensitivity of monoclonal antibodies. To obtain B-lymphocytes producing high-quality specific antibodies for cell fusion during the immunization phase of the antigen, we prepared a TH2-Cell stimulatory delivery system as a novel adjuvant. Astragalus polysaccharide has a good ability to enhance antigenic immune response, and it was encapsulated in biocompatible materials PLGA as an immunostimulatory factor to form the delivery system (APS-PLGA). The preparation conditions of APSP were optimized using RSM to attain the highest utilization of APS. Immunization against ZEN-BSA antigen using APSP as an adjuvant to obtain B lymphocytes producing ZEN-specific antibodies for cell fusion. As results present, APSP could induce a stronger TH2 immune response through differentiating CD4 T cells and promoting IL-4 and IL-6 cytokines. Moreover, it could slow down the release efficiency of ZEN-BSA and enhance the targeting of ZEN-BSA to lymph nodes in vivo experiments. Ultimately, the sensitivity of mouse serum ZEN-specific antibodies was enhanced upon completion of immunization, indicating a significant upregulation of high-quality B lymphocyte expression. In the preparation of monoclonal antibodies, the proportion of positive wells for the first screening was 60%, and the inhibition rates of the antibodies were all similar (>50%). Then we obtained the ZEN monoclonal antibody with IC50 of 0.049 ng/mL, which was more sensitive than most antibodies prepared under conventional adjuvants. Finally, a TRFIAS strip assay was preliminarily established with a LOD value of 0.246 ng/mL.

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.

Abstract LB247: TCPAplus: An LLM-empowered chatbot for analyzing a large protein expression atlas of human cancers

Abstract We present a comprehensive functional proteomics resource comprising nearly 9,000 samples from The Cancer Genome Atlas and Cancer Cell Line Encyclopedia, using ~500 high-quality antibodies through reverse phase protein arrays. To enhance its utility, we have developed an analytical chatbot harnessing advanced large language models. This intuitive platform empowers researchers to effortlessly query protein-centered cancer omics data, conduct related analyses, and visualize results using natural language, significantly easing the translation from intricate proteogenomic data to valuable biological insights. Citation Format: Wei Liu, Jun Liu, Yitao Tang, Chaozhong Liu, Meiyi Song, Zhenlin Ju, Shweth V. Kumar, Yiling Lu, Rehan Akbani, Gordon Mills, Han Liang. TCPAplus: An LLM-empowered chatbot for analyzing a large protein expression atlas of human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB247.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

MCSFR+ monocytes/macrophages are activated to produce IL-4/13 in the gill lamellae of grass carp after infection with bacterial pathogens

Macrophage colony-stimulating factor receptor (MCSFR) is a cell lineage marker for monocytes/macrophages and has been applied for cell typing in mammals. However, lack of high-quality monoclonal antibodies hampers isolation and functional characterization of monocytes/macrophages in fish. A monoclonal antibody (mAb) against the extracellular region of grass carp (Ctenopharyngodon idella, Ci) MCSFR using the recombinant protein produced in the E. coli cells was generated in this study. Western blotting showed the mAb could recognize the native CiMCSFR protein derived from tissues and the recombinant CiMCSFR protein expressed in the E. coli cells and HEK293 cells. The antibody did not cross-react with the MCSFR homolog in zebrafish, indicating that it has good species specificity. Immunofluorescence analysis showed MCSFR+ monocytes/macrophages distributed in the gills, hindgut, head kidney and spleen of healthy grass carp. Furthermore, Aeromonas hydrophila infection resulted in increased MCSFR+ monocytes/macrophages in the lamina propria and epithelium of hindgut mucosa. In addition, Flavobacterium columnare infection led to the infiltration of MCSFR+ monocytes/macrophages in the gills, and interestingly, the MCSFR+ monocytes/macrophages in the gill lamellae but not filaments are activated to produce IL-4/13A and IL-4/13B, while MCSFR+ monocytes/macrophages in the hindgut did not produce IL-4/13A and IL-4/13B. Our results indicate that monocytes/macrophages are participated in mediating mucosal immune responses to bacterial infections and will help understand host immune defense to bacterial diseases.

Abstract P17: Epigenomic fingerprinting of limited primary immune cells using automated CUT&RUN

Abstract Chromatin structure drives gene expression programs during immune cell development and in cancer, making it central to the advancement of precision medicine. Indeed, the study of chromatin regulation has provided valuable insight on effector cell differentiation and function, anti-tumor immune responses, and therapeutic resistance. Epigenomic features – such as histone post-translational modifications (PTMs) and chromatin-associated proteins – mark distinct genomic compartments (e.g., promoters, enhancers) and regulate chromatin function/gene expression. Mapping the genomic distribution of these may uncover new biomarkers or drug targets and provides a rich context for exploring immunology. Despite widespread interest, integration of epigenomics in large-scale immunotherapy and/or drug research has been hampered by the poor sensitivity, high background, and low throughput of traditional chromatin mapping technologies, most notably ChIP-seq. Instead, efforts to characterize chromatin dynamics have focused on DNA methylation (bisulfite-seq) or chromatin accessibility (ATAC-seq). However, these assays provide an incomplete view of the chromatin landscape, limiting their ability to define cell differentiation pathways or derive mechanistic insight into disease etiology. Here, we present autoCUT&RUN, a high-throughput assay for rapid, ultra-sensitive profiling of epigenomic features from FACS-isolated primary cells or tissues. This workflow generates reliable profiles from ~10,000 cells per reaction, and is supported by a rigorous optimization strategy, high-quality antibodies, and quantitative spike-in controls. As part of a multi-site collaboration with the Immunological Genome Consortium, we used our autoCUT&RUN platform to build a comprehensive epigenomic database of primary mouse immune cells - composed of >1,500 epigenomic profiles from >100 different FACS-isolated immune cell types. These studies set the stage to leverage high-throughput epigenomics in immunotherapy and precision medicine applications. Citation Format: Ellen N. Weinzapfel, Keith E. Maier, Matthew R. Marunde, Vishnu U. Sunitha Kumary, Carolina P. Lin Windham, Danielle N Maryanski, Liz Albertorio-Saez, Dughan J. Ahimovic, Michael J. Bale, Juliana J. Lee, Bryan J. Venters, Michael-Christopher Keogh, Immunological Genome Consortium. Epigenomic fingerprinting of limited primary immune cells using automated CUT&RUN [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P17.

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

Generation of a highly specific and potent antibody against Ce-lamin/LMN-1.

High quality antibodies are useful tools for research in cell and developmental biology. We have obtained highly specific and potent guinea pig-derived polyclonal antibodies against Ce-lamin/ LMN-1 . Western blotting experiments using these antibodies demonstrated that maternally loaded Ce-lamin/ LMN-1 protein is very stable, can perdure through embryonic and larval development and remain detectable in homozygous null lmn-1 mutant adults.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

A guide to selecting high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence.

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.

Novel hapten design, highly sensitive monoclonal antibody production, and immunoassay development for rapid screening of illegally added chloramphenicol in cosmetics

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5–10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics.

Identification of high-performing antibodies for Rab1A and Rab1B for use in Western Blot, immunoprecipitation and immunofluorescence

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.

Automated Cut&run Brings Scalable Epigenomic Profiling to Hematology

Genome-wide association studies (GWAS) and corresponding transcriptomics research have been leveraged to identify disease risk variants in hematological disorders with the hopes of developing more personalized treatment regimens. Interestingly, >90% of GWAS-identified variants are found in non-coding regions of the genome, indicating they likely affect the regulatory machinery that governs chromatin structure. Non-coding elements, such as enhancers, play important roles in supporting chromatin mechanisms, including cell-type specific gene expression, cell fates and disease states. However, these variants are difficult to study compared to protein-coding mutations and have been challenging to directly associate with phenotypic readouts. To date, efforts to understand the effects of non-coding variants on chromatin dynamics have been focused on chromatin accessibility (ATAC-seq) and DNA methylation profiling. Notably, these assays can only provide a binary, “open-or-closed,” view of chromatin and often fail to provide mechanistic insight into disease etiology. Epigenomic features - such as histone post-translational modifications (PTMs) and chromatin-associated proteins - mark distinct genomic compartments (e.g., promoters, enhancers) and regulate chromatin structure, gene expression, and cell function. Mapping these features provides a rich context to study cell fate and has great potential for discovering new biomarkers and drug targets. Despite this, efforts to integrate epigenomics into large scale lymphatic and myeloid research have been hampered by the poor sensitivity, high background, and low throughput of traditional chromatin mapping technology (i.e., ChIP-seq). Here, we present autoCUT&RUN, a high throughput assay for rapid, ultra-sensitive profiling of epigenomic features from FACS-isolated primary cells or tissues. This workflow generates reliable profiles from <10,000 cells per reaction, and is supported by a rigorous optimization strategy, high-quality antibodies, and quantitative spike-in controls. As part of a multi-site collaboration with the Immunological Genome Consortium, we used our autoCUT&RUN platform to build a comprehensive epigenomic database of mouse immune cells - composed of >1,500 epigenomic profiles from >100 different FACS-isolated primary immune cell types. These studies set the stage to leverage high-throughput epigenomics for multi-site clinical studies to identify and/or characterize novel biomarkers for precision medicine applications. Further, we will discuss our integration of CUT&RUN and Enzymatic Methylation-sequencing (CUT&RUN-EM) to deliver a true multiomic view of the co-occurrence of epigenomic features and DNA methylation, which can reveal multivalent cellular mechanisms and deconvolute sample heterogeneity.

Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence.

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.