177 results about "Protease digestion" patented technology

Methods, compositions, kits, and apparatus are provided wherein the aminoacyl-tRNA synthetase system is used to analyze amino acids. The method allows very small devices for quantitative or semi-quantitative analysis of the amino acids in samples or in sequential or complete proteolytic digestions. The methods can be readily applied to the detection and / or quantitation of one or more primary amino acids by using cognate aminoacyl-tRNA synthetase and cognate tRNA. The basis of the method is that each of the 20 synthetases and / or a tRNA specific for a different amino acid is separated spatially or differentially labeled. The reactions catalyzed by all 20 synthetases may be monitored simultaneously, or nearly simultaneously, or in parallel. Each separately positioned synthetase or tRNA will signal its cognate amino acid. The synthetase reactions can be monitored using continuous spectroscopic assays. Alternatively, since elongation factor Tu:GTP (EF-Tu:GTP) specifically binds all AA−tRNAs, the aminoacylation reactions catalyzed by the synthetases can be monitored using ligand assays. Microarrays and microsensors for amino acid analysis are provided. Additionally, amino acid analysis devices are integrated with protease digestions to produce miniaturized enzymatic sequenators capable of generating either N- or C-terminal sequence and composition data for a protein or peptide. The possibility of parallel processing of many samples in an automated manner is discussed.

A method for quantitating vitamin D metabolites directly in blood plasma or serum, without the need for prior purification of the vitamin D metabolites, comprising a digestion of the serum proteins with a serine protease such as proteinase K and sequence of steps for inhibiting the proteinase K activity in the competitive binding analysis. The advantages of this method are its high accuracy over the whole range of physiologically relevant values and that it can be easily adapted for a fully automated analysis of serum and plasma samples.

It is an object of the invention to provide novel approaches capable of detecting activated protease and also detecting protease activation on real time at a high sensitivity in a noninvasive manner. By the method for detecting activated protease of the invention, an indicator in a circular form comprising the C-half fragment of luciferase (Luc-C) and the N-half fragment of luciferase (Luc-N) linked together through a substrate peptide for a protease is introduced in an in vitro assay system or in cells. Upon digestion of the substrate peptide by the protease, Luc-N and Luc-C together reconstructs active luciferase, so that the activated protease can be detected by assaying the luminescence signal from the luciferase.

The invention discloses an albumen microsphere conjugate for detecting acrosin activity and a preparation method and application of the albumen microsphere conjugate. The albumen microsphere conjugate is characterized in that monodisperse micron microspheres obtained by combining fluorescein and the acrosome reaction substrate, namely zona pellucida protein are prepared, the acrosin activity is detected by aid of change in fluorescence intensity, and the specific steps include preparation of monodisperse microspheres modified through surface carboxylation or amination, surface modificaction, fluorescence labeling of protein polypeptide, conjugation of fluorescent protein polypeptide and functional microspheres and detection of the acrosin activity. The albumen microsphere conjugate has the advantages that protease digestion on the microspheres is realized through the polymer microsphere technology and the protein labeling and conjugating technology, and enzymatic activity is evaluated; the albumen microsphere conjugate is mainly applied to flow detection of the acrosin activity, synchronous detection of an acrosome reaction and the acrosin activity can be realized, and acrosome functions can be evaluated more truthfully and accurately.

An albumin denaturing agent for digesting an albumin by a protease efficiently is provided. The albumin denaturing agent contains quaternary ammonium having a hydrocarbon group with a carbon number of 12 or more, or a salt of the quaternary ammonium. The albumin in a sample is digested by the protease in the presence of the albumin denaturing agent, a glycated part of the thus obtained albumin digestion product and a FAOD effect a reaction, and a redox reaction between the glycated part and the FAOD is measured, thereby determining a ratio (GA (%)) of the glycated albumin of the glycated albumin with respect to the albumin.

The present invention provides for novel reagents whose fluorescence increases in the presence of particular proteases. The reagents comprise a characteristically folded peptide backbone conjugated to two fluorophores such that the fluorophores are located opposite sides of a cleavage site. When the folded peptide is cleaved, as by digestion with a protease, the fluorophores provide a high intensity fluorescent signal at a visible wavelength. Because of their high specificity and their high fluorescence signal in the visible wavelengths, these protease indicators are particularly well suited for detection of protease activity in biological samples, in particular in frozen tissue sections. Thus this invention also provides for methods of detecting protease activity in situ in frozen sections.

The invention relates to a bifid yeast fermentation composition and a preparation method thereof. The fermentation composition is formed by mixing bifid yeast lysis substance mother liquor and filtrate according to a certain ratio. The preparation method comprises the following steps of: S1: carrying out anaerobic culture and activation to prepare a first-level fermentation seed solution; S2: inoculating the first-level fermentation seed solution into a micro-aerobic fermentation solid culture medium to obtain a second-level fermentation seed solution; S3: transferring the second-level fermentation seed solution into the fermentation culture medium to carry out micro-aerobic fermentation; S4: through fermentation broth centrifugation, separating bifid yeast filtrate from bifidobacterium bodies; S5: after the bifidobacterium bodies are resuspended and broken, obtaining the bifid yeast lysis substance mother liquor; and S6: mixing the bifid yeast filtrate with the lysis substance mother liquor to obtain a bifid yeast fermentation product. Since bifidobacterium longum is used for carrying out multilevel anaerobic / micro-aerobic fermentation, the problem of a microorganism pollution influence is restricted, and safety is further improved; indexes, including an oxygen content, are controlled in a step-by-step fermentation process, and each physicochemical index of a final product is stable; and through a compound protease digestion technology, the content of polypeptides under same-scale bifidobacterium fermentation is effectively improved.

The invention discloses a nucleic acid rapid-purifying method and kit. Specifically, the present invention discloses a reagent combination used for rapid purification of nucleic acids from a nucleic acid-containing sample, the kit prepared on the basis of the reagent combination, and the nucleic acid rapid-purifying method on the basis of the reagent combination or the kit. The method of the invention has the advantages of high nucleic acid yield, high nucleic acid purity, rapidness, room temperature operation, no need of protease digestion process and the like, and is suitable for analysis or extraction and purification of the nucleic acids of various downstream experiments.

The invention discloses a novel preparation technology of targeting antitumor fusion protein (lipid peroxidation). The novel preparation method comprises the steps of building a fusion expression protein which takes the glutathione S-transferase A (TrxA)-His-tag (His6-Tag)-SUMO protease recognition substrate as a protein soluble expression auxiliary fragment and takes LHRH (luteinizing hormone releasing hormone)-PEA (phenylethylamine) trans-cell penetrating peptides-ONC (LPO for short) as a target segment, connecting the two proteins with each other by a correct read frame and carrier positive sequence and converting to enter into expression bacteria, finally building fusion protein which is connected with six expression substances in series, crudely extracting, carrying out metal chelating medium purification in the presence of imidazole, and carrying out SUMO protease digestion. Compared with the LPO prepared by a conventional method, the LPO prepared by the novel preparation technology disclosed by the invention can obviously improve the inhibiting effect on the tumor cell lines such as colon cancer HT-29 cells, ovarian cancer OVCAR3 cells, cervical adenocarcinoma HeLa cells and liver cancer HepG-2 cells.

An adjustable, low mass-to-charge (m / z) filter is disclosed employing electrospray ionization to block ions associated with unwanted low m / z species from entering the mass spectrometer and contributing their space charge to down-stream ion accumulation steps. The low-mass filter is made by using an adjustable potential energy barrier from the conductance limiting terminal electrode of an electrodynamic ion funnel, which prohibits species with higher ion mobilities from being transmitted. The filter provides a linear voltage adjustment of low-mass filtering from m / z values from about 50 to about 500. Mass filtering above m / z 500 can also be performed; however, higher m / z species are attenuated. The mass filter was evaluated with a liquid chromatography-mass spectrometry analysis of an albumin tryptic digest and resulted in the ability to block low-mass, “background” ions which account for 40-70% of the total ion current from the ESI source during peak elution.

The present invention relates to a method for phosphorylation site-specific labeling of phosphoproteome with a site-specific tagging reagent and analyzing of the resulting labeled one, more especially, a method for in-situ tagging of phosphorylation sites of phosphoproteins retained in polymeric gel with a nucleophilic tagging reagent. It also relates a method for generating new proteolytic cleavable sites at formerly phosphorylation sites by a proper choice of a nucleophilic tagging reagent. It also relates to a method for phosphopeptides analysis and phosphorylation site identification by in-gel digestion of the previously in-gel tagged proteins and subsequent mass analysis of the resulting peptides. The invention provides in-gel chemical tagging method for phosphoaminoacid residue of phosphoproteins retained in polymeric gel matrix. Phosphoprotein can be immobilized into gel matrix by a variety of methods such as gel electrophoresis. The immobilized phosphoproteins are retained in gel matrix during tagging reaction to phosphorylated aminoacid residue of phosphoproteins, and the resulting tagged proteins are also retained in gel matrix till following purification steps like washing of the tagging reagents are accomplished. The tagged proteins is digested by protease, and the resulting digested peptides is released from gel into solution and applied for peptide mass analysis.

Compositions and methods for treating or preventing, for example, overweight or obesity are provided. Compositions provided comprise zinc-charged, protease digested serum or milk proteins. Compositions are administered in a therapeutically effective amount to treat or prevent, for example, overweight or obesity.