This service exclusively searches for literature that cites resources. Please be aware that the total number of searchable documents is limited to those containing RRIDs and does not include all open-access literature.
The affinity of an antibody for its antigen serves as a critical parameter for antibody evaluation. The evaluation of antibody-antigen affinity is essential for a successful antibody-based assay, particularly immunoprecipitation (IP), due to its strict dependency on antibody performance. However, the determination of antibody affinity or its quantitative determinant, the dissociation constant (Kd), under IP conditions is difficult. In the current study, we used a NanoLuc-based HiBiT system to establish a HiBiT-based quantitative immunoprecipitation (HiBiT-qIP) assay for determining the Kd of antigen-antibody interactions in solution. The HiBiT-qIP method measures the amount of immunoprecipitated proteins tagged with HiBiT in a simple yet quantitative manner. We used this method to measure the Kd values of epitope tag-antibody interactions. To accomplish this, FLAG, HA, V5, PA and Ty1 epitope tags in their monomeric, dimeric or trimeric form were fused with glutathione S-transferase (GST) and the HiBiT peptide, and these tagged GST proteins were mixed with cognate monoclonal antibodies in IP buffer for the assessment of the apparent Kd values. This HiBiT-qIP assay showed a considerable variation in the Kd values among the examined antibody clones. Additionally, the use of epitope tags in multimeric form revealed a copy number-dependent increase in the apparent affinity.
In the past few decades, numerous approaches have been developed to investigate protein-protein and protein-nucleic acid interactions (PPIs and PNIs). Affinity purification methods such as co-immunoprecipitation (co-IP) are commonly used to detect and isolate the macromolecular complexesresulting from these interactions. In this article, we describe a two-step co-immunoprecipitation (TIP) technique. As compared to standard co-IP, TIP provides increased specificity in the isolation of PPIs or PNIs under native expression conditions, dramatically reducing the abundance of nonspecific binders and thus facilitating downstream analyses of the interaction complexes. Here, we report a detailed TIP procedure that we used to purify a protein-protein complex from Burkitt lymphoma cells and from primary human CD4+ T cells. In addition, this unit describes an application of TIP for the isolation of transcription-factor-bound chromatin. © 2018 by John Wiley & Sons, Inc.
Native immunoprecipitation followed by protein A-mediated recovery of the immuno-complex is a powerful tool to study protein-protein interactions. A limitation of this technique is the concomitant recovery of large amounts of immunoglobulin, which interferes with down-stream applications such as mass spectrometric analysis and Western blotting. Here we report a detergent-based "soft" elution protocol that allows effective recovery of immunoprecipitated antigen and binding partners, yet avoids elution of the bulk of the immunoglobulin.
The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a "one size fits all" rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM) [an acceptable in vitro model for primary, human macrophage cells], we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10min. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge.
The transcriptional regulatory network in prokaryotes controls global gene expression mostly through transcription factors (TFs), which are DNA-binding proteins. Chromatin immunoprecipitation (ChIP) with DNA sequencing methods can identify TF binding sites across the genome, providing a bottom-up, mechanistic understanding of how gene expression is regulated. ChIP provides indispensable evidence toward the goal of acquiring a comprehensive understanding of cellular adaptation and regulation, including condition-specificity. ChIP-derived data's importance and labor-intensiveness motivate its broad dissemination and reuse, which is currently an unmet need in the prokaryotic domain. To fill this gap, we present proChIPdb (prochipdb.org), an information-rich, interactive web database. This website collects public ChIP-seq/-exo data across several prokaryotes and presents them in dashboards that include curated binding sites, nucleotide-resolution genome viewers, and summary plots such as motif enrichment sequence logos. Users can search for TFs of interest or their target genes, download all data, dashboards, and visuals, and follow external links to understand regulons through biological databases and the literature. This initial release of proChIPdb covers diverse organisms, including most major TFs of Escherichia coli, and can be expanded to support regulon discovery across the prokaryotic domain.
Advanced technologies are producing large-scale protein-protein interaction data at an ever increasing pace. A fundamental challenge in analyzing these data is the inference of protein machineries. Previous methods for detecting protein complexes have been mainly based on analyzing binary protein-protein interaction data, ignoring the more involved co-complex relations obtained from co-immunoprecipitation experiments.
Protein-protein interactions in cells are widely explored using small-scale experiments. However, the search for protein complexes and their interactions in data from high throughput experiments such as immunoprecipitation is still a challenge. We present "4N", a novel method for detecting protein complexes in such data. Our method is a heuristic algorithm based on Near Neighbor Network (3N) clustering. It is written in R, it is faster than model-based methods, and has only a small number of tuning parameters. We explain the application of our new method to real immunoprecipitation results and two artificial datasets. We show that the method can infer protein complexes from protein immunoprecipitation datasets of different densities and sizes.
We report on a detailed chromatin immunoprecipitation (ChIP) protocol for the unicellular green alga Chlamydomonas reinhardtii. The protocol is suitable for the analysis of nucleosome occupancy, histone modifications and transcription factor binding sites at the level of mononucleosomes for targeted and genome-wide studies. We describe the optimization of conditions for crosslinking, chromatin fragmentation and antibody titer determination and provide recommendations and an example for the normalization of ChIP results as determined by real-time PCR.
With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. Chromatin immunoprecipitation (ChIP-seq) is the omics technique that enables genome wide localization of transcription factor (TF) binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP-target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach for finding TF-binding sites in ChIP-seq, with roots in digital signal processing that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq. Ritornello is freely available at https://github.com/KlugerLab/Ritornello.
Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.
HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure two consensus sequences for HMGA2 binding have been identified.
Chromatin immunoprecipitation (ChIP) analysis is widely used to identify the locations in genomes occupied by transcription factors (TFs). The approach involves chemical cross-linking of DNA with associated proteins, fragmentation of chromatin by sonication or enzymatic digestion, immunoprecipitation of the fragments containing the protein of interest, and then PCR or hybridization analysis to characterize and quantify the genomic sequences enriched. We developed a computational model of quantitative ChIP analysis to elucidate the factors contributing to the method's resolution. The most important variables identified by the model were, in order of importance, the spacing of the PCR primers, the mean length of the chromatin fragments, and, unexpectedly, the type of fragment width distribution, with very small DNA fragments and smaller amplicons providing the best resolution of TF binding. One of the major predictions of the model was also validated experimentally.
The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.
S-glutathionylation is a reversible post-translational modification of proteins that generate a mixed disulfide between glutathione to thiolate anion of cysteine residues in target proteins. In the last ten years, S-glutathionylation has been extensively studied since it represents the cellular response to oxidative stress, in physiological as well as pathological conditions. This modification may be a protective mechanism from irreversible oxidative damage and, on the other hand, may modulate protein folding and function. Due to the importance of S-glutathionylation in cellular redox signaling, various methods have been developed to identify S-gluthationylated proteins. Herein, we describe two easy methods to recognized S-glutathionylation of a target protein after oxidative stress in cellular extracts based on different immunoprecipitation procedures. The immunoprecipitation assay allows the capture of one glutathionylated protein using a specific antibody that binds to the target protein. The presence of S-glutathionylation in the immunoprecipitated protein is identified using anti-glutathione antibody. The second type of approach is based on the detection of the glutathionylated protein with biotin/streptavidin technique. After different steps of protection of non-oxidized thiolic groups and reduction of S-glutathionylated groups, the newly-formed protein free-thiols are labeled with biotin-GSH. The modified protein can be isolate with streptavidin-beads and recognized using an antibody against target protein. •S-glutathionylation is a reversible post-translational modification of proteins that recently has been emerged as important signaling in the redox regulation of protein function.•Both methods to identify glutathionylated proteins are economic, easy and do not require particular equipment.•The setups of both methods guarantee high reproducibility.
Development of reliable, quantitative technologies for serodiagnosis of Toxoplasma gondii infection remains desirable. The luciferase immunoprecipitation system (LIPS) is a relatively simple, highly sensitive, and rapid quantitative immunoassay. The major advantages of this assay over ELISA are a wider dynamic range, shorter overall assay time, and less sample volume. In this study, we aimed to use this method for the serodiagnosis of toxoplasmosis. Recombinant Toxoplasma antigens (dense granule antigens GRA6, GRA7, and GRA8 and bradyzoite antigen BAG1) fused with nanoluciferase (Nluc, a small luciferase enzyme) were expressed in Escherichia coli, purified, and tested in LIPS assays with sera from experimental mice infected with T. gondii and a WHO standard anti-Toxoplasma human immunoglobulin (TOXM). In the experimentally infected mice, LIPS assays detected antibodies against Nluc-GRA6, Nluc-GRA7, and Nluc-GRA8 as early as day 14, whereas antibodies against Nluc-BAG1 remained undetected until day 21 and then showed significant elevation on day 60. In TOXM sera, LIPS assays with each Nluc recombinant protein produced reliable standard curves with a coefficient of determination (R2) of 0.980-0.989 for GRA6, 0.986-0.990 for GRA7, 0.998-0.999 for GRA8, and 0.942-0.987 for BAG1. The detection limits were estimated to be 3.9, 2, 1, and 1 IU/ml for rGRA6, rGRA7, rGRA8, and rBAG1, respectively. The LIPS assay for toxoplasmosis could detect antibodies against T. gondii in the mouse and human sera with a reasonably high sensitivity. We consider the LIPS assay to be a promising alternative tool for screening, diagnosing, and monitoring toxoplasmosis. In particular, detection of antibodies against BAG1 may be useful for a longitudinal seroprevalence study in suspected high-risk areas on the basis of its elevated serum concentration in the chronic phase.
Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. However, non-biased methods to identify molecules bound to specific genomic loci in vivo are limited. Here, we applied insertional chromatin immunoprecipitation (iChIP) to direct identification of components of insulator complexes, which function as boundaries of chromatin domain. We found that the chicken β-globin HS4 (cHS4) insulator complex contains an RNA helicase protein, p68/DDX5; an RNA species, steroid receptor RNA activator 1; and a nuclear matrix protein, Matrin-3, in vivo. Binding of p68 and Matrin-3 to the cHS4 insulator core sequence was mediated by CCCTC-binding factor (CTCF). Thus, our results showed that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP.
The small ubiquitin-like modifier (SUMO) is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM). Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SUMOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2) is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP) for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chromatin factors at specific genomic regions.
G quadruplex structures play an important role in regulating DNA replication and transcription and mRNA translation. Although there are several techniques that can determine its formation in vitro, the study of RNA G quadruplexes in vivo is not simple. In the current protocol, we describe an optimized technique (RNA G quadruplex immunoprecipitation [rG4IP]) to selectively pull down native cytoplasmic RNAs containing G quadruplex structures in mammalian cells. We also use a bicistronic plasmid to confirm and pinpoint the structure location. For complete details on the use and execution of this protocol, please refer to Surani et al. (2022).
Ten-eleven translocation (TET) proteins, encoding dioxygenase for DNA hydroxymethylation, are important players in nervous system development and disease. In addition to their proverbial enzymatic role, TET proteins also possess non-enzymatic activity and function in multiple protein-protein interaction networks, which remains largely unknown during oligodendrocyte differentiation. To identify partners of TET1 in the myelinating cells, we performed proteome-wide analysis using co-immunoprecipitation coupled to mass spectrometry (IP-MS) in purified oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes (mOLs), respectively. Following a stringent selection of MS data based on identification reliability and protein enrichment, we identified a core set of 1211 partners that specifically interact with TET1 within OPCs and OLs. Analysis of the biological process and pathways associated with TET1-interacting proteins indicates a significant enrichment of proteins involved in regulation of cellular protein localization, cofactor metabolic process and regulation of catabolic process, et al. We further validated TET1 interactions with selected partners. Overall, this comprehensive analysis of the endogenous TET1 interactome during oligodendrocyte differentiation suggest its novel mechanism in regulating oligodendrocyte homeostasis and provide comprehensive insight into the molecular pathways associated with TET1.
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method designed to detect interactions between chromatin and the proteins bound to it. This method has been widely used for characterizing epigenetic landscapes in many cell types; however, a limiting factor has been the requirement of a high number of cells. Here, we describe a protocol for ChIP in epidermal cells from a newborn mouse, purified by fluorescence-activated cell sorting (FACS). This protocol has been optimized specifically for prefixed, low cell numbers, resulting in enough immunoprecipitated DNA suitable for genome-wide analysis.
Welcome to the FDI Lab - SciCrunch.org Resources search. From here you can search through a compilation of resources used by FDI Lab - SciCrunch.org and see how data is organized within our community.
You are currently on the Community Resources tab looking through categories and sources that FDI Lab - SciCrunch.org has compiled. You can navigate through those categories from here or change to a different tab to execute your search through. Each tab gives a different perspective on data.
If you have an account on FDI Lab - SciCrunch.org then you can log in from here to get additional features in FDI Lab - SciCrunch.org such as Collections, Saved Searches, and managing Resources.
Here is the search term that is being executed, you can type in anything you want to search for. Some tips to help searching:
You can save any searches you perform for quick access to later from here.
We recognized your search term and included synonyms and inferred terms along side your term to help get the data you are looking for.
If you are logged into FDI Lab - SciCrunch.org you can add data records to your collections to create custom spreadsheets across multiple sources of data.
Here are the facets that you can filter your papers by.
From here we'll present any options for the literature, such as exporting your current results.
If you have any further questions please check out our FAQs Page to ask questions and see our tutorials. Click this button to view this tutorial again.
Year:
Count: