Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specific granules are argentafugic when ultrathin sections of Araldite-embedded atria are stained according to the periodic acid-thiocarbohydrazide-silver proteinate technique of Thiery. The entire core of the atrial specific granules is moderately positive after ultrathin sections of glutaraldehyde-fixed, glycol methacrylate- (GMA-) embedded atria are stained with phosphotungstic acid at a low pH. A similar reaction is shown by the cell coat, intercalated discs, residual bodies (C-granules), and Z-discs, as well as by a very small portion of the Golgi complex. Analogous results are obtained with semithin sections of GMA- embedded atria stained according to the periodic acid-Schiff (PAS) technique. In ultrathin sections of GMA-embedded atria stained with dialyzed colloidal iron (DI), the cell coat of the cardiocytes is positive, unlike all the other cytoplasmic organelles. When ultrathin sections of GMA-embedded atria are incubated with proteolytic enzymes (pronase, pepsin, or trypsin), atrial specific granules and Z-bands and, to a much lesser degree, cell coat and sarcolemma are selectively digested. Proteins are also distinctly demonstrated in the paranuclear specific granules by a variety of histochemical techniques. These results indicate that atrial specific granules are rich in proteins and possess a weak complement of complex carbohydrates.
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PMID:Chemical nature of atrial specific granules. 5 70

We have examined the possibility of improving the present methods of detecting bromodeoxyuridine (BrdU) and for combining the PAS reaction with the BrdU detection by means of immunogold-silver staining (IGSS). This was done in testes fixed in Carnoy or Bouin, and in parts of the small intestine which were fixed in Carnoy or periodate-lysine-paraformaldehyde (PLP). All tissues were embedded in a mixture of glycol methacrylate and butanediol-monoacrylate. It was found to be impossible to carry out BrdU detection using HCl hydrolysis and trypsin digestion in combination with a PAS reaction. However, incubation of the plastic sections in periodic acid for a period of 30 minutes appeared to make it possible to eliminate the HCl denaturation step and to carry out a specific PAS reaction. Moreover, after incubation in periodic acid, trypsin digestion was no longer required to make the BrdU label accessible in GMA-embedded sections, nor to re-expose the antigenic sites in plastic sections of tissues fixed with cross-linking fixatives. In this way the loss of cell structures, which is inevitable when trypsin is used, can be avoided. Now a BrdU detection with improved morphology can be combined with the PAS reaction in the same plastic section in order to stain tissue carbohydrates. This is important for tumour diagnosis, where the PAS reaction can be very useful.
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PMID:Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold--silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction. 131 38

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.
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PMID:Antigen localization in immunoperoxidase-stained plastic-embedded soft tissues. 245 78

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.
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PMID:Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections. 618 93

In formalin-fixed, paraffin-embedded tissue enhanced or de novo immunostaining can be obtained by microwave boiling of sections in a metal salt or buffer solution. In this paper this new technique is reviewed and important factors influencing final results are discussed. Microwave antigen retrieval can also be applied for immunohistochemistry on plastic GMA sections. Here the action of the microwave method is probably mainly due to breaking the bonds between GMA and proteins prohibiting immunostaining. The microwave methods do not require trypsin treatment.
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PMID:Notes on the application of microwaves for antigen retrieval in paraffin and plastic tissue sections. 839 49

The immobilization of trypsin on porous glycidyl methacrylate (GMA-GDMA) beads has been investigated. In particular, the distribution within the beads of trypsin and of dextran used for hydrophilizing the bead surface prior to protein immobilization was investigated with confocal microscopy. For the system investigated, the fluorescence intensity profiles obtained when using borate buffer as an ambient solution displayed a distinct minimum at the center of the beads, irrespective of the observation depth. However, by reduction of the refractive index difference between the solution and the beads through the addition of glucose to the aqueous solution, artifacts relating to optical length differences could be reduced. For both low molecular weight fluorescein isothiocyanate (FITC), FITC-labeled trypsin, and FITC-labeled dextran, an essentially homogeneous distribution throughout the beads was observed. This simple "contrast matching" method seems therefore to be an interesting tool when investigating the distribution of immobilized protein in porous chromatography media. Copyright 1999 Academic Press.
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PMID:Confocal Microscopy Studies of Trypsin Immobilization on Porous Glycidyl Methacrylate Beads. 1060 63

The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads.
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PMID:Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization. 1091 49

Rigid porous poly(glycidyl methacrylate-divinylbenzene) (P(GMA-DVB)) microspheres were synthesized through suspension polymerization with a mixture of isooctane and 4-methyl-2-pentonal as the porogen. The microspheres were intended to use as column packing materials for protein separation. However, irreversible adsorption of protein was found on the polymer microsphere. To circumvent the problem, polyethylene glycol (PEG) was coupled to the microspheres. The coupling reaction took place between the hydroxyl group of PEG and the epoxy group of the P(GMA-DVB) solid medium in the presence of boron trifluoride. The density of PEG immobilized onto the P(GMA-DVB) can be determined easily by saponification of modified microsphere firstly and then titration of glycerol-PEG. The effect of the cross-linker content of microsphere on the density of PEG immobilization was investigated. Molecular weight of PEG was found to influence the PEG-immobilization density, which subsequently affects the hydrophilicity of the modified P(GMA-DVB). Bovine serum albumin (BSA) and trypsin were used as model proteins to examine the adsorption and desorption properties of the modified P(GMA-DVB) microspheres. The results demonstrated that P(GMA-DVB) porous microsphere with 20% DVB and modified with PEG4000 showed excellent adsorption and desorption properties. Adsorption capacity of BSA on the modified microsphere attained to 51.6 mg/g microsphere, and BSA mass recovery and trypsin activity recovery was up to 97.6% and 98.7%, respectively. The modified microsphere was demonstrated to be a promising hydrophobic interaction chromatography material for purification of protein.
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PMID:Modification of poly(glycidyl methacrylate-divinylbenzene) porous microspheres with polyethylene glycol and their adsorption property of protein. 1682 38

Fabrication of poly(glycidyl methacrylate-co-ethylene dimethacrylate) [also referred to as poly(GMA-co-EDMA)] monoliths was optimized as supporting material for trypsin digestion nanoreactors. Reaction parameters, such as polymerization time, porogen concentration, and monomer to crosslinker ratios, were evaluated in respect to the permeability of the resulting monolith and their effect on digestion efficiency, estimated by mass spectrometric analysis of a model protein cytochrome C. The structural homogeneity of the resulting monolithic support was checked by scanning electron microscopy. The best nanoreactor performance, measured by the reduction of nanoreactor backpressure and increased sequence coverage of cytochrome C, was achieved with 8% 2-octanol (porogen) 20%/20% glycidyl methacrylate to ethylene glycol dimethacrylate ratio and 5 h of polymerization time. Digestion of as low as 3 microg of cytochrome C with 77% sequence coverage was obtained using the optimized trypsin nanoreactor.
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PMID:Optimization of poly(GMA-co-EDMA) monolithic support for trypsin nanoreactor fabrication. 1955 52

Trypsin was immobilized on glycidylmethacrylate-co-divinylbenzene (GMA/DVB) polymerized in pipet tips for online enzymatic digestion of proteins. The major advantages of in-tip digestion are easy handling and small sample amount required for analysis. Microwave-assisted digestion was applied for highly efficient and time saving proteolysis. Adaption to an automated robotic system allowed fast and reproducible sample treatment. Investigations with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography coupled with electrospray-ionization mass spectrometry (LC-ESI/MS) attested high sequence coverages (SCs) for the three standard proteins, myoglobin (Myo, 89%), bovine serum albumin (BSA, 78%) and alpha-casein (alpha-Cas, 83%). Compared to commercially available trypsin tips, clear predominance concerning the digestion performance was achieved. Storageability was tested over a period of several weeks and results showed only little decrease (<5%) of protein sequence coverages. The application of microwave-assisted in-tip digestion (2 min) with full automation by a robotic system allows high-throughput analysis (96 samples within 80 min) and highly effective proteolysis.
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PMID:Ultrafast microwave-assisted in-tip digestion of proteins. 1963 39


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