Chemically modified porous silica gel as a bioadsorbent and a biocatalyst.
TL;DR: Epoxy silica gel, prepared by reaction of porous silica Gel with γ-glycidoxypropyltriethoxysilane, was coupled with m -aminobenzamidine and the resulting matrix was used for trypsin purification.
Abstract: Aminopropyl silica gel was prepared from porous silica gel by reaction with γ-aminopropyltrimethoxysilane in toluene and was used for immobilizing chymotrypsin (EC 3.4.4.5) and human serum albumin. Immobilized chymotrypsin was used for the resolution of N -acetyl- dl -phenylalanine and immobilized human serum albumin was used for the purification of goat anti-human serum albumin. Epoxy silica gel, prepared by reaction of porous silica gel with γ-glycidoxypropyltriethoxysilane, was coupled with m -aminobenzamidine and the resulting matrix was used for trypsin purification.
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TL;DR: It was found that treatment of gels with dithiothreitol prior to impregnation with silver nitrate results in more reproducible staining patterns that are also qualitatively similar to those obtained with Coomassie blue.
Abstract: The rapid, ultrasensitive silver stains that have been developed recently for detecting proteins in polyacrylamide gels show variation in staining from gel to gel and do not stain certain proteins at all. It was found that treatment of gels with dithiothreitol prior to impregnation with silver nitrate results in more reproducible staining patterns that are also qualitatively similar to those obtained with Coomassie blue. In addition, it obviates the need for treatment with intense light, and results in sensitivities at least as high as those obtained with previously published methods.
3,999 citations
TL;DR: The AMPPD technique was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total, and proved extremely sensitive for immunoblotting.
Abstract: The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.
93 citations
TL;DR: The first two non-silanizing coupling methods are simple, inexpensive and non-hazardous compared to the third, more complex method in which an initial Correspondance: to PVS.
Abstract: Several methods for immobilizing anti-T2 mycotoxin monoclonal antibodies on quartz fibers, for use in optical sensor development, have been evaluated with respect to the surface density and stability of the immobilized proteins. the first method activates matrix hydroxyl groups using p-toluenesulfonyl chloride (TSC). the second method activates these groups using p-nitrophenyl chloroformate (NPCF). the third method requires an initial silanization using 3-aminopropyltriethoxysilane (APTES) followed by carrier activation with glutaraldehyde. the activated carrier in all three methods is then reacted with the amino groups of the protein. the first two non-silanizing coupling methods are simple, inexpensive and non-hazardous compared to the third, more complex method in which an initial Correspondance: to PVS
36 citations
TL;DR: A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings and the obtained results showed good correlation with the hospital immunoturbidimetric reference method.
Abstract: A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings. Polyclonal rabbit anti-human albumin was used as a capture antibody and monoclonal mouse anti-human albumin as a detection antibody in a two-site immunometric assay requiring no additional washing procedures. The polyclonal anti-human albumin was conjugated to silica microparticles (solid phase) and the monoclonal antibody to dextran-coated nanoscaled superparamagnetic particles (tracer). Quantification of human albumin in undiluted urine was performed by adding 2 microL urine to a measuring vial containing solid-phase, superparamagnetic tracer and reaction buffer and then inverting the vial by hand for 20 s. The measuring vial was allowed to stand for 6 min prior to detection, in order for the solid-phase sediment to form at the bottom of the vial. Lastly, the measuring vial was placed into a magnetic permeability detector, which measured the enrichment of superparamagnetic tracer in the sediment due to complex formation with human albumin. Total analysis time was 6.5 min. A linear response was obtained for 0-400 mg/L albumin with a detection limit of 5 mg/L. The total coefficient of variation (CV) was 11% calculated from four consecutive runs on a urine sample containing 11.1 mg/L human albumin during 3 consecutive days. Human urinary albumin analysis was performed on 149 patient samples using the MIA technique and the obtained results showed good correlation with the hospital immunoturbidimetric reference method (y = 1.004x + 4.01, R2 = 0.978, N = 149) and a commercially available point of care albumin analysis provided by HemoCue Inc. (y = 0.98x + 5.8, R2 = 0.833, N = 90).
29 citations
TL;DR: The environmental parameters necessary for increased production of mature, 35‐kDa active protease in strains of L. monocytogenes are described and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease is described.
Abstract: Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.
25 citations
Cites methods from "Chemically modified porous silica g..."
...Prior to coupling of bacitracin to the silica, the latter was activated by the addition of an epoxy group (Roy and Kundu 1979; van den Burg et al. 1989)....
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TL;DR: The ultra-violet inactivation of lysozyme has been studied and found to conform to a first order reaction, and the relation of molecular weight to quantum yield, as well as the other findings, are discussed.
Abstract: 1. 1. A rapid and reproducible method for measurement of lysozyme activity is described. 2. 2. Using this method, the ultra-violet inactivation of lysozyme has been studied and found to conform to a first order reaction. 3. 3. The measured quantum yield for inactivation at 2537 A is 0.024 and remains unaltered over the pH range 3.6 to 12.0. A small effect of concentration on quantum yield is indicated. 4. 4. Inactivation is accompanied by photo-oxidation and other secondary processes, with no apparent liberation of free amino acids or peptides. 5. 5. The relation of molecular weight to quantum yield, as well as the other findings, are discussed.
1,041 citations
TL;DR: It has been demonstrated that carbohydrate bonded supports may be used in the chromatography of proteins, nucleic acids, and polysaccharides.
Abstract: Glycerolpropylsilane bonded phases have been found to control the adsorption and/or denaturation of proteins and nucleic acids on controlled porosity glass supports. The bonded-phase thickness is 18-19A while the amount of glycerol moiety varies from 80 to 150 mumoles/g depending on support pore diameter. It has been demonstrated that carbohydrate bonded supports may be used in the chromatography of proteins, nucleic acids, and polysaccharides.
278 citations
132 citations
TL;DR: Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration and the values of the inhibition constant, K i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer forThrombin than for trypsIn.
Abstract: Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, Ki, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low Ki values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.
91 citations
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