38 results about "Affinity label" patented technology

A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such labels are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilization of the fusion protein.

Analytical reagents and mass spectrometry-based methods using these reagents for the rapid, and quantitative analysis of proteins or protein function in mixtures of proteins. The methods employ affinity labeled protein reactive reagents having three portions: an affinity label (A) covalently linked to a protein reactive group (PRG) through a linker group (L). The linker may be differentially isotopically labeled, e.g., by substitution of one or more atoms in the linker with a stable isotope thereof. These reagents allow for the selective isolation of peptide fragments or the products of reaction with a given protein (e.g., products of enzymatic reaction) from complex mixtures. The isolated peptide fragments or reaction products are characteristic of the presence of a protein or the presence of a protein function in those mixtures. Isolated peptides or reaction products are characterized by mass spectrometric (MS) techniques. The reagents also provide for differential isotopic labeling of the isolated peptides or reaction products which facilitates quantitative determination by mass spectrometry of the relative amounts of proteins in different samples. The methods of this invention can be used for qualitative and quantitative analysis of global protein expression profiles in cells and tissues, to screen for and identify proteins whose expression level in cells, tissue or biological fluids is affected by a stimulus or by a change in condition or state of the cell, tissue or organism from which the sample originated.

The invention provides methods and compositions for azide tagging of biomolecules. In one embodiment of the invention, proteins are tagged by metabolic incorporation of prenylated azido-analog substrates. Examples of such analogs are azido farnesyl diphosphate and azido farnesyl alcohol. The azido moiety in the resulting modified proteins provides an affinity tag, which can be chemoselectively captured by an azide-specific conjugation reaction, such as the Staudinger reaction, using a phosphine capture reagent. When the capture agent is biotinylated, the resulting conjugates can be detected and affinity-purified by streptavidin-linked- HRP and streptavidin-conjugated agarose beads, respectively. The invention allows detection and isolation of proteins with high yield, high specificity, and low contamination without harsh treatment of proteins.

A method for selective labeling of phosphate groups in natural and synthetic oligomers and polymers in the presence of chemically related groups such as carboxylic acid groups. The method is specifically applicable to biological oligomers and polymers, including phosphopeptides, phosphoproteins and phospholipids. In a specific embodiment, selective labeling of phosphate groups in proteins and peptides, for example, facilitates separation, isolation and detection of phosphoproteins and phosphopeptides in complex mixtures of proteins. Selective labeling can be employed to selectively introduce phosphate labels at phosphate groups in an oligomer or polymer, e.g., in a peptide or protein. Detection of the presence of the label, is used to detect the presence of the phosphate group in the oligomer or polymer. The method is useful for the detection of phosphoproteins or phosphopeptides. The phosphate label can be a colorimetric label, a radiolabel, a fluorescent or phosphorescent label, an affinity label or a linker group carrying a reactive group (or latent reactive group) that allows selective attachment of the oligomer or polymer (protein or peptide) to a phosphate label, to an affinity label or to a solid support. The method can be combined with well-known methods of mass spectrometry to detect and identify phosphopeptides and phosphoproteins.

The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

The invention provides a divinyl sulfamide linker as well as preparation and an application thereof. The divinyl sulfamide linker has the structural formula shown in the formula I or the formula II. One end of the novel divinyl sulfamide linker can be coupled with a polypeptide or protein, and the other end can be connected with an affinity tag or a tracing fluorescent substance or an active drug.

The invention generally relates to compositions for maximizing capture of affinity-labeled molecules on solid supports. The disclosed methods and compositions were developed to maximize depletion of ribosomal RNA from total RNA samples, which is useful to improve the quality of RNA preparations used for applications such as massively parallel sequencing. The RNA depletion method is based on using long affinity-labeled RNA molecules that are complementary to all or part of the target ribosomal RNAs, as subtractive hybridization probes.

The invention discloses an isoandrographolide photoaffinity labeling molecular probe. The structure of the molecular probe is represented in a formula (I), wherein n1 ranges from 1 to 5 and n2 ranges from 0 to 2. Furthermore, the invention discloses a preparation and a pharmaceutical composition of the isoandrographolide photoaffinity labeling molecular probe. The compound, which is excellent in TNF-alpha inhibitory activity, can be used for researching and discovering target protein of isoandrographolide having an action of regulating macrophage function, and the compound, as a tool medicine, can be used for medicine development of a diagnostic reagent of the target protein as well as a regulator thereof.

The invention relates to a light affinity labelling small molecular probe based on maslinic acid and preparation method thereof, belonging to the technical field of organic compound and preparation thereof. Natural product maslinic acid is taken as initial raw material, compound I is obtained under the protection of acetyl, the compound I reacts with oxalyl chloride to obtain compound II, the compound II reacts with ethane diamine under single protection of Boc to obtain compound III, the compound III firstly strips Boc protecting group in HCl ethyl acetate solution and then reacts with segment IV with light affinity labelling group to obtain compound V, the compound V strips acetyl protecting group by hydrolysis with presence of alkali, so as to obtain light affinity labelling small molecular probe VI. The probe molecule can be used for researching and discovering active compound maslinic acid in vivo action target, action mode of target spot and structural information of active site and provides important information for research and development of related medicine.

The present invention relates to a method for production and purification of affinity tagged macromolecular complexes, such as ribosomes. More closely, the method comprises in-frame fusion of a nucleotide sequence specific for an affinity tag and a selection marker, wherein the fusion is at the chromosomal site of a gene encoding a multicopy protein, and wherein the macromolecular complex is expressed with multiple copies of said affinity tag. The invention also relates to affinity tagged ribosomes, to cells comprising such affinity tagged ribosomes, and to various uses thereof.

A process for synthesizing and separating secretory IgA from a mixture of IgA monmer and IgA dimer is provided. The process includes covalently binding affinity tagged or epitope tagged recombinant secretory component to the IgA dimer in the mixture and binding the affinity tagged or an epitope tagged secretory IgA to immobilized moieties on the solid phase support resin to which the affinity tag or epitope tag binds and then eluting the affinity tagged or an epitope tagged secretory IgA with release buffer. A process for synthesizing and separating secretory IgM from a mixture of IgM and other plasma proteins is also provided. The process includes covalently binding affinity tagged or an epitope tagged recombinant secretory component to the IgM in the mixture and binding the affinity tagged or an epitope tagged secretory IgM to immobilized moieties on the solid phase support resin and then eluting the peptide tagged secretory IgM with a release buffer.

Novel withanolide chemical genetic probes identify the in vivo binding target of withaferin A, which is the intermediate filament type III protein vimentin. In addition, a withanolide-based small molecule screening method screens drug candidates that target intermediate filament type III proteins. The method includes introducing a tagged linker covalently bonded to the withanolide molecule to form a withanolide probe. Better or alternative small molecule compounds as potential drug candidates can be generated based on their likely affinity for the determined binding site in vimentin. The affinity labeled withanolide can also be used to find intermediate filament-associated proteins using chemical proteomics by extracting proteins from cells that were exposed to withanolide-biotin analog. The withanolide probes can be used to monitor expression of vimentin, in tumor samples or other diseased tissues. Withaferin analogs can be used as a treatment for diverse vimentin-associated disorders, such as cancers, angiofibrotic diseases, and chronic inflammation.

This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Methods are described for incorporating N-terminal, C-terminal and (optionally) affinity markers into a nascent protein

Methods for producing isotopically-labelled peptides are provided. Aspects of the method include: contacting a sample including a metabolically tagged protein with a cleavable probe to produce a probe-protein conjugate; separating the probe-protein conjugate from the sample; digesting the probe-protein conjugate to produce a probe-peptide conjugate; and cleaving a cleavable linker to release an isotopically labelled peptide. The method may further include: identifying a predetermined isotopic pattern in a mass spectrum; determining an amino acid sequence of the isotopically labelled peptide; and identifying the site of protein glycosylation based on the determined amino acid sequence. Also provided are cleavable probes for practicing the subject methods, described by the Formula: A-L-(M-Z) where A is an affinity tag, L is a cleavable linker, M is an isotopic label and Z is a chemoselective tag capable of cross-linking a metabolically tagged protein. Compositions and kits for practicing the subject methods are also provided.

The invention provides a novel method for synthesizing hyperpolarized xenon-129 (HP Xe) biosensors by using pseudo-rotaxane structures of gamma-cyclodextrin. These supramolecular complexes form novel ternary structures in the presence of HP Xe which can be detected via 129Xe MR spectroscopy and imaging techniques. The rotaxane-type complex can be tagged with an affinity label for detecting a target in a biological subject.

The invention provides a preparation method of an organophosphorus degrading enzyme-based multifunctional catalyst, the organophosphorus degrading enzyme-based multifunctional catalyst and applicationof the organophosphorus degrading enzyme-based multifunctional catalyst. The preparation method comprises the following steps: a composite yolk shell structure nano material is directly added into crude enzyme liquid of organophosphorus degrading enzyme with an affinity label, mixed and separated, and then the organophosphorus degrading enzyme-based multifunctional catalyst is obtained. The preparation method of the organophosphorus degrading enzyme-based multifunctional catalyst is easy to operate, the requirement for the enzyme purity is low, the carrier can be directionally combined with the enzyme, the prepared organophosphorus degrading enzyme-based multifunctional catalyst is low in cost, an organophosphorus pesticide can be detected, the organophosphorus pesticide can be further degraded in a cascade mode, and a final product, namely p-aminophenol has important application value.

The invention provides a divinyl sulfamide linker as well as preparation and an application thereof. The divinyl sulfamide linker has the structural formula shown in the formula I or the formula II. One end of the novel divinyl sulfamide linker can be coupled with a polypeptide or protein, and the other end can be connected with an affinity tag or a tracing fluorescent substance or an active drug.

The present invention relates to compositions, methods for expressing, and related methods for purifying calreticulin that is free of an affinity label or tag (i.e., non-tagged calreticulin). The invention provides useful methods for commercial production of human calreticulin in a bacterial expression system such as Escherichia coli.

Provided are a target region enrichment method based on multiplex PCR, and a reagent, the method comprising: connecting a first linker and a second linker respectively at two ends of a nucleic acid segment containing target regions to be enriched so as to obtain a linker-connected product; performing a PCR amplification on the linker-connected product using a first primer specifically bound to the first linker and a second primer specifically bound to the second linker to obtain an amplified product, the first primer or the second primer having a first affinity label; capturing a single strand having the first affinity label in the amplified product using a solid phase carrier; performing single primer linear amplification using a third primer with the captured single strand as a template; performing exponential amplification using the third primer and the first primer, with the linearly amplified product as the template, to obtain a product containing the target regions.

The invention relates to a phospholipid nano-disk chromatography medium and a preparation method and application thereof. The phospholipid nano-disk chromatography medium comprises a charged phospholipid nano disk and an affinity matrix; the charged phospholipid nano disk comprises phospholipid and a film scaffold protein with an affinity label; the charged phospholipid nano disk is connected withan affinity matrix through the affinity label. The phospholipid nano-disk chromatography medium has both binding capacity and protection capacity to film proteins, so that the actual system containing the film proteins can be purified and reconstructed on a chromatography column in one step; and the phospholipid nano-disk chromatography medium has reversibility and regeneration performance; the phospholipid nano-disk chromatography medium solves the problems of serious protein activity loss, long and complicated steps and time-consuming and labor-consuming operation in the traditional film protein research technology, is simple and efficient to operate, improves the film protein activity recovery rate by more than 20 times, and shortens the treatment time by more than 95%.

The invention generally relates to compositions for maximizing capture of affinity-labeled molecules on solid supports. The disclosed methods and compositions were developed to maximize depletion of ribosomal RNA from total RNA samples, which is useful to improve the quality of RNA preparations used for applications such as massively parallel sequencing. The RNA depletion method is based on using long affinity-labeled RNA molecules that are complementary to all or part of the target ribosomal RNAs, as subtractive hybridization probes.

The invention relates to a method for utilizing ubiquitin-binding domain (UBD) fusion protein to separate and detect ubiquitination protein. The principle is that a fusion protein is constructed by using an affinity label and a ubiquitin-binding domain. The fusion protein can be expressed in colon bacillus, purified and fixed on a gel particle through the ligand of the affinity label. Mixed culture is carried out on the gel particle with the ubiquitin-binding domain fusion protein and lysate of cell or tissue, and the ubiquitination protein in the lysate can be separated out through precipitation. On the aspect of the detection of ubiquitination protein, the method disclosed by the invention is higher in efficiency, detection capacity and flexibility compared with the current antibody immune precipitation detection method. Moreover, on the aspect of economic benefit, the cost of detecting ubiquitination protein in the invention is extremely lower than that of expensive antibody immune precipitation method; and the method can be applied to fundamental research of protein ubiquitination and the detection of ubiquitination protein in medical diagnosis test.

The present invention relates to compositions, methods for expressing, and related methods for purifying calreticulin that is free of an affinity label or tag (i.e., non-tagged calreticulin). The invention provides useful methods for commercial production of human calreticulin in a bacterial expression system such as Escherichia coli.

The invention relates to a phospholipid nano-disk chromatography medium and a preparation method and application thereof. The phospholipid nano-disk chromatography medium comprises a charged phospholipid nano disk and an affinity matrix; the charged phospholipid nano disk comprises phospholipid and a film scaffold protein with an affinity label; the charged phospholipid nano disk is connected withan affinity matrix through the affinity label. The phospholipid nano-disk chromatography medium has both binding capacity and protection capacity to film proteins, so that the actual system containing the film proteins can be purified and reconstructed on a chromatography column in one step; and the phospholipid nano-disk chromatography medium has reversibility and regeneration performance; the phospholipid nano-disk chromatography medium solves the problems of serious protein activity loss, long and complicated steps and time-consuming and labor-consuming operation in the traditional film protein research technology, is simple and efficient to operate, improves the film protein activity recovery rate by more than 20 times, and shortens the treatment time by more than 95%.

Provided is a compound. According to the embodiment of the invention, the invention relates to the compound represented by the formula (I), or stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs of the compound represented by the formula (I). The compound can effectively obtain two or three or more ubiquitin chains and ubiquitin modifiedsubstrates; moreover, the compound can be used for modifying polypeptide or protein side chains with affinity labels, fluorescent groups, PEG or fatty hydrocarbons and the like; and the compound can play an important role in design of polypeptide drugs, biochemical testing, functional optimization and the like.

Provided is a compound. According to the embodiment of the invention, the invention relates to the compound represented by the formula (I), or stereoisomers, tautomers, nitrogen oxides, solvates, metabolites, pharmaceutically acceptable salts or prodrugs of the compound represented by the formula (I). The compound can effectively obtain two or three or more ubiquitin chains and ubiquitin modifiedsubstrates; moreover, the compound can be used for modifying polypeptide or protein side chains with affinity labels, fluorescent groups, PEG or fatty hydrocarbons and the like; and the compound can play an important role in design of polypeptide drugs, biochemical testing, functional optimization and the like.

Systems and methods of coding for and isolating a fusion protein are provided. The fusion protein may be expressed by a DNA sequence and comprise an adenylate kinase linked by its carboxy-terminus to a biologically active polypeptide or protein. Later processing steps include isolating the biologically active polypeptide or protein via affinity elution of the fusion proteins with substrate analog of adenylate kinase.

The present invention discloses one kind of ligand fixing mode for the streptavidin coated chip (SA-chip) surface in BIAcore biosensor. In traditional SA-chip, the ligand is fixed by means of the specific mutual action between biotin and streptavidin, and the very powerful mutual action makes the SA-chip become disposable commodity. Instead, the present invention fuses the affinity label and the target ligand by means of the mutual action between streptavidin and affinity label to realize the directional fixing of target ligand. Under mild controllable condition, the chip surface may be regenerated for reuse of the SA-chip. The new ligand fixing process results in greatly lowered experiment cost and possesses high application value.