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Results for “immunoprecipitation”

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PubMed2024

Phage Immunoprecipitation and Sequencing-a Versatile Technique for Mapping the Antibody Reactome.

Characterizing the antibody reactome for circulating antibodies provide insight into pathogen exposure, allergies, and autoimmune diseases. This is important for biomarker discovery, clinical diagnosis, and prognosis of disease progression, as well as population-level insights into the immune system. The emerging technology phage display immunoprecipitation and sequencing (PhIP-seq) is a high-throughput method for identifying antigens/epitopes of the antibody reactome. In PhIP-seq, libraries with sequences of defined lengths and overlapping segments are bioinformatically designed using naturally occurring proteins and cloned into phage genomes to be displayed on the surface. These libraries are used in immunoprecipitation experiments of circulating antibodies. This can be done with parallel samples from multiple sources, and the DNA inserts from the bound phages are barcoded and subjected to next-generation sequencing for hit determination. PhIP-seq is a powerful technique for characterizing the antibody reactome that has undergone rapid advances in recent years. In this review, we comprehensively describe the history of PhIP-seq and discuss recent advances in library design and applications.

Humans↗

PubMed1978

Immunoprecipitation of a cytoplasmic precursor of rat-liver cytochrome oxidase.

A simple method for the isolation of rat liver cells is described. The cells are shown, by an isotope dilution method, to maintain a constant rate of protein synthesis for 8 h of incubation. Antibodies to purified rat liver cytochrome oxidase were raised in rabbits and used to investigate the labeling of cytochrome oxidase in isolated rat liver cells and in vivo. The data demonstrate the occurrence of a precursor of the subunits of cytochrome oxidase that are synthesized in the cytoplasm. 1. Dodecylsulfate gel electrophoresis of the immunoprecipitates from isolated rat liver cells that had been labeled with [35S]methionine for 1 h showed a single radioactive peak with a molecular weight of 50000. 2. Judged by the effects of cycloheximide and chloramphenicol the labeled protein is synthesized on cytoplasmic ribosomes. 3. After labeling for 1 h in vivo with [3H]leucine the labeled protein appears to be exclusively associated with the hepatic microsomal fraction. 4. Ouchterlony double-diffusion analysis demonstrated immunological relationship between the precipitates from microsomes and cytochrome oxidase. In addition to the precipitates derived from mitochondria and microsomes immunoprecipitates were also obtained from the cytosol in comparable amounts; these again were immunologically related. The occurrence of large amounts of precursor(s) (or degradation products) of cytochrome oxidase in rat liver fractions is interpreted in terms of a regulatory pool for amino acid homeostasis in the organism.

Animals↗

PubMed1979

Human cytomegalovirus-induced immediate early antigens: analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation.

Immediate early antigen (IEA) induced in human lung fibroblasts by human cytomegalovirus was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation with IEA-positive human sera. Two polypeptides of 76,000 daltons (76K) and 82K were detectable within 90 min after infection. Polypeptides of similar molecular weight were also found in immunoprecipitates of human cytomegalovirus-infected cells nonpermissive for virus replication. IEA is located within the nucleus, although some of the 76K material appears to be located on the outer nuclear membrane. Raising salt concentrations in the extraction buffer increased antigen extraction. The contribution of these IEA polypeptides to IEA nuclear fluorescent staining is discussed.

Animals↗

PubMed1976

Yeast cytochrome c messenger RNA. In vitro translation and specific immunoprecipitation of the CYC1 gene product.

An assay based upon indirect immunoprecipitation has been developed for yeast cytochrome c and apocytochrome c. The specificity of this assay was demonstrated by its ability to selectively precipitate cytochrome c from an autolysate of yeast cell proteins. Translation of the polypeptide chain of cytochrome c in a wheat germ extract programmed with yeast poly(A) RNA was demonstrated using this immunoprecipitation assay. Translation of poly(A) RNA from yeast strains carrying nonsense mutations in the cyc1 gene yielded in vitro cytochrome c polypeptides which were shorter than the wild type protein by the amount expected for polypeptide chains which had terminated at the nonsense codon. The in vivo rate of cytochrome c synthesis was shown to be 6-fold greater in derepressed cells than in glucose-repressed cells. The 6-fold difference is sufficient to account for the 6-fold higher level of cytochrome c in derepressed than in repressed cells. The level of translatable cytochrome c mRNA is at least 4 times as high in derepressed as in glucose-repressed cells, suggesting that regulation occurs at some step in the synthesis of this messenger.

Cytochrome c Group↗

PubMed1978

[Indirect immunoprecipitation by rat liver polyribosomes using antibodies to tyrosine aminotransferase].

A fraction of rat liver polyribosomes is isolated, which in its immunochemical characteristics considerably enriched with polyribosomes capable to synthesize hydrocortisone-induced liver tyrosine aminotransferase isoenzyme. This specific polyribosome fraction was purified by immunochemical fractionation of total liver polyribosomes using indirect precipitation. The content of polyribosomes in immunoprecipitates comprise 0.4-0.8% of its initial amount (before immunochemical fractionation). The ratio of specific polyribosomes in immunoprecipitates varies from 20 to 45%, which corresponds to 25-100-fold purification. The data obtained suggest that the method of indirect precipitation can be an efficient step in the isolation procedure of individual mRNA.

Animals↗

PubMed2025

Chromatin Immunoprecipitation for Standard, Rare, or Weakly Binding Proteins.

Various proteins interact with specific genome regions, playing crucial roles in gene regulation. Chromatin Immunoprecipitation (ChIP) is the most commonly used method to study protein-DNA interactions in vivo. By combining ChIP with high-throughput sequencing, ChIP-seq allows for studying the genome-wide localization of proteins. Although several ChIP protocols are available for plant tissues, they are primarily designed for histone modifications and abundant proteins with high DNA-binding affinity, which are considered as the "standard targets." Here we describe a ChIP protocol for plant tissues not only optimized for the standard targets but also adapted for proteins with low abundances or weak DNA-binding ability. Successful execution of the protocol enables reliable generation of DNA templates for quantitative PCR or libraries for next-generation sequencing, which makes it an effective tool for analyzing genomic interactions of a wide range of proteins.

Chromatin Immunoprecipitation↗

PubMed2026

A guide to selecting high-performing antibodies for TMEM175 (UniProt ID: Q9BSA9) for use in western blot, immunoprecipitation, and immunofluorescence.

TMEM175 is the pore-forming subunit of a lysosomal K+ channel complex that regulates lysosomal pH stability and membrane potential. To further investigate its cellular functions and implications in neurodegenerative diseases, antibody reagents are needed. Here we have characterized six TMEM175 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.

Humans↗

PubMed2026

A guide to selecting high-performing antibodies for DJ-1 ( PARK7) (Q99497) for use in western blot, immunoprecipitation, and immunofluorescence.

DJ-1 is a multifunctional protein that plays a pivotal role in cellular protection against oxidative stress and neurodegeneration. Mutations in the PARK7 gene are associated with early-onset familial Parkinson's disease. Here we have characterized sixteen DJ-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. 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 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.

Protein Deglycase DJ-1↗

PubMed2026

A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence.

Syntenin-1 is the Syndecan-binding protein 1 and a PDZ domain-containing adaptor protein that regulates diverse cellular processes through its interactions with transmembrane receptors, cytoskeletal components, and signaling molecules. Here we have characterized twelve Syntenin-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. 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 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.

Syntenins↗

PubMed2026

A guide to selecting high-performing renewable antibodies for KIF5A (Q12840) across western blot, immunoprecipitation, and immunofluorescence.

Kinesin Family Member 5A (KIF5A) is an important protein for anterograde cellular transport. We characterized ten commercially available research antibodies against KIF5A for use in western blot, immunoprecipitation, and immunofluorescence using a standardized workflow. Antibody performance was assessed by comparing signal in a wild-type cell line and its corresponding knockout derivative. This work is part of a broader collaborative public-good initiative to improve biomedical research by systematically evaluating commercial antibodies against human proteins and openly sharing the data as a resource for the scientific community. We encourage readers to use this report as a guide for selecting antibodies best suited to their specific applications.

Kinesins↗

PubMed1977

Immunoprecipitation of herpes simplex virus type 1 antigens with different antisera and human cerebrospinal fluids.

Rabbit convalescent and hyperimmune sera, human patient and blood donor sera, as well as cerebrospinal fluids of humans with herpes simplex virus encephalitis all recognize similar major antigenic components in herpes simplex virus infected rabbit or human cells as shown by electrophoretic analysis of immunoprecipitates. Besides the main glycoproteins with an apparent molecular weight of 100,000 (peak I) the antisera precipitate glycoproteins in a region of an apparent mol. wt. of 60,000--80,000 (peak II), which were resolved into distinct glycoprotein species only by antibody-containing cerebrospinal fluids. The peak II glycoproteins appear on the surface of the infected cell early, and absorb neutralizing antibodies, whereas the peak I glycoproteins are less accessible. Both antigens can be demonstrated in the cell as early as about 2 hours post infection. All major antigenic components studied were found to be glycosylated except one protein with an apparent mol. wt. of 110,000. The herpesvirus specificity of these antigens is demonstrated by a variety of control experiments. The antigens detected are virion components.

Animals↗

PubMed1977

Correlation of apparent molecular weight and antigenicity of viral proteins: an SDS-page separation followed by acrylamide-agarose electrophoresis and immunoprecipitation.

A simple method is described which combines a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE) in the first demension with a second electrophoresis, at right angles to the first, into an agarose matrix. The proteins, separated by SDS-PAGE, are exposed to appropriate antisera after the second stage electrophoresis and immunoprecipitates form in the agarose corresponding to the relative electrophoretic mobilities of proteins in the first stage SDS-PAGE separation. The method thus provides a simple, reproducible means for correlating antigenicity with apparent molecular weight of proteins. The technique is qualtitative, but requires smaller quantities of antisera than more conventional immunoelectrophoretic methods such as rocket electrophoresis.

Antigens, Viral↗

PubMed1978

An immunoprecipitation-dissociation technique for large scale antibody purification and an antigen consumption electroimmunoassay for antibody quantitation. A model study with antibodies to pregnancy zone protein.

A simple immunoprecipitation--dissociation technique for large scale purification of antibodies is described, which comprises selective denaturation of the antigen and recovery of the antibody fraction by exclusion chromatography at low pH. Its use is illustrated by the purification of antibodies to pregnancy zone protein. A purification factor of about 60 was achieved. An antigen consumption electroimmunoassay was also developed which permits quantitative determination of the antigen binding activity of antibodies with a given specificity. The methods have general application.

Antibodies↗

PubMed1979

An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates.

Polyoma T antigen immunoprecipitates contain a protein kinase-like activity which preferentially phosphorylates material of 50-60,000 daltons molecular weight. Phosphorylation is not diminished in extracts of polyoma tsA mutant-infected cells shifted to the nonpermissive temperature late in infection, conditions which inactivate the large T antigen. Phosphorylation is reduced or absent in cells infected with polyoma host range nontransforming (hr-t) mutants, which have defective small and medium T antigens. The major acceptor of phosphate is not the heavy chain of immunoglobulin, but appears to be the polyoma medium T antigen. The large T antigen is also phosphorylated, but usually to a lower specific activity. In terms of acid and alkali sensitivity and electrophoretic and chromatographic mobility in one and two dimensions, the phosphorylated residue behaves identically to phosphotyrosine and differently than phosphorylated serine, threonine, lysine and histidine.

Animals↗

PubMed2025

Utility of Plasma Cell-free Chromatin Immunoprecipitation to Detect Cardiac Allograft Rejection.

BACKGROUND: Antibody-mediated rejection (AMR) remains the major risk factor for allograft loss across all solid organ transplantation. Unfortunately, its diagnosis relies on biopsy, an invasive gold standard that often sample unaffected allograft tissue leading to missed diagnosis. Plasma donor-derived cell-free DNA (dd-cfDNA) is noninvasive biomarker that has high sensitivity but low specificity for AMR diagnosis. This proof-of-concept study assessed the utility of cell-free chromatin immunoprecipitation (cfChIP) as a surrogate for gene expression to detect cardiac AMR and the associated pathobiology. METHODS: The discovery GRAfT multicenter cohort of heart transplant patients (NCT02423070) identified AMR, acute cellular rejection (ACR), and stable controls based on biopsy and ddcfDNA results. Plasma cfChIP-sequencing was performed to identify peaks, associated genes and pathobiological pathways. Plasma from an external cohort (GTD, NCT01985412) was also analyzed to verify pathways identified. Digital droplet PCR (ddPCR) assays targeting differential regions were constructed to test the diagnostic performance of cfDNA to detect AMR/ACR from stable controls (rejection-specific assays) or AMR from ACR (AMR-specific assays). RESULTS: The cohort included 21 AMR, 28 ACR, and 45 stable controls from GRAfT and GTD, and 23 healthy controls. cfChIP detected expected active genes, including housekeeping genes and gene targets of transplant immunosuppressive drugs but not inactive genes. Unsupervised clustering of the discovery GRAfT cohort assigned 95% of samples correctly as AMR, ACR or stable control. Differential analysis identified pathobiological pathways of AMR such as neutrophil degranulation and complement activation. The pathways were consistent in GTD samples. Rejection-specific assays detected AMR/ACR from controls with AUC of 0.78 - 0.95. AMR-specific assays detected AMR from ACR with AUC of 0.71 - 0.85, sensitivities of 0.73 - 0.94 and specificities of 0.73 - 0.80. CONCLUSION: This study provides valuable preliminary data supporting the use of cfChIP to detect AMR and the associated pathobiological pathways.

Allograft rejection↗

PubMed2026

Detecting Meiotic Crossing-Overs in Maize Using Chromatin Immunoprecipitation-Sequencing (ChIP-seq).

During meiosis, homologous chromosomes engage in reciprocal exchanges of segments in a process known as crossing over (CO). About 85% of CO events in maize are products of the class I pathway. Class I COs are interference-sensitive, meaning that the formation of one CO reduces the likelihood of another CO forming close by. This protocol describes a chromatin immunoprecipitation-sequencing (ChIP-seq)-based method for mapping meiotic COs in maize, using an antibody against MutL Homolog 3 (MLH3), a key component of the class I CO pathway. CO sites are determined by Illumina sequencing of DNA isolated from MLH3-associated chromatin fragments. Traditionally, COs have been identified through genetic mapping, which relies on the segregation of genetic markers in the progeny of hybrid plants. However, conventional genetic mapping provides limited resolution and requires large numbers of progeny individuals. The MLH3 ChIP-seq approach enables direct detection of COs, providing high-resolution and genome-wide coverage, including genome regions with low DNA sequence polymorphism, which are inaccessible to genetic CO mapping. Furthermore, MLH3 ChIP-seq enables screening of thousands of CO events, greatly accelerating the analysis and reducing its cost. This protocol can also be used to examine any chromatin-bound meiotic proteins and adapted to studying chromatin-associated proteins in somatic cells.

Journal Article↗

PubMed1979

Immunoglobulins G, A and M in normal and pathologic human sera determined with the spot immunoprecipitate assay (SIA) in multi-assay plates.

A simple rapid spot immunoprecipitate assay (SIA) is presented. The antigen--antibody reaction takes place in a field of alternating current; small amounts of specific antisera are used, and the influence of diffusion or electromobility of the antigen is avoided. Determinations of IgG, IgA and IgM in sera from healthy individuals correlated highly significantly with radial immunodiffusion (RID) results. The reaction conditions in SIA, particularly the availability of a sufficiency of specific antibodies, may permit more accurate estimates of monoclonal forms of Ig than in RID, where the combination depends on diffusion of one reactant. The precision of SIA quantitation of IgG and IgA was within +/- 10% and +/- 13% for IgM. Working ranges of the standard curves were from about 25--150 ng for G and A and 75--300 ng for IgM; least squares regression coefficients for four sets of standards on three SIA plates were 0.995 for IgG, 0.969 for IgA, and 0.986 for IgM. IgA at 7.5 ng in a 2.5 mg/l dilution was at the lower limit of demonstrability. Photometric registrations and estimates made by visual inspection compared highly significantly (P less than 0.001), differing by +/- 3%.

Antigen-Antibody Reactions↗

PubMed1979

Quantification of the pregnancy-specific beta1-glycoprotein (SP1) by immunoprecipitation techniques: the influence of a cross-reacting high molecular weight alpha2-protein.

Two serum proteins with SP1 determinants were separated by means of size chromatography and preparative zone electrophoresis. One of the proteins had beta1 electrophoretic mobility and an estimated molecular weight of 90,000, and the other had alpha2-mobility and a molecular weight around 400,000. When available antibody preparations to SP1 were used, the high molecular weight protein formed immunoprecipitates only when polyethyleneglycol (PEG) was added to the agarose. It is shown that the high molecular weight protein has a pronounced effect on quantification of SP1 by rocket immunoelectrophoresis, whereas the effect was less pronounced but still considerable when quantification was performed by radial immunodiffusion.

Antigens↗