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PubMedDate not supplied

Integrated removal of nucleic acids and recovery of LDH from homogenate of beef heart by affinity precipitation.

Lactate dehydrogenase (LDH) was purified from beef heart homogenate by affinity precipitation. The protein purification was integrated with nucleic acid removal and was done by precipitation of nucleic acids by addition of poly(ethylene imine) PEI onto which a ligand, Cibacron blue, had been coupled. The yield of LDH after elution from the precipitate was 63%, the purification factor 6.9 and the nucleic acid content was reduced by 98%. The capacity of the affinity polymer Cibacron blue-PEI is dependent on the nucleic acid concentration in the homogenate. The beef heart homogenate had an unfavourable ratio of nucleic acids to LDH. Precipitation with recirculated Cibacron blue-PEI, already complexed with some nucleic acids, improved the yield of the enzyme to 74%. The loss of Cibacron blue-PEI, when recirculated, was less than 1% after each cycle.

Animals↗

PubMed2000

Recovery of a monoclonal antibody from hybridoma culture supernatant by affinity precipitation with Eudragit S-100.

An IgG1 monoclonal antibody (MAB) was isolated from hybridoma culture supernatant by affinity precipitation with an Eudragit S-100-based heterobifunctional ligand. Affinity binding was performed in a homogeneous aqueous phase at pH 7.5 followed by precipitation of the bound affinity complex by lowering the pH to 4.8. After two washing steps, elution of specifically bound MAB was achieved by incubating the precipitate with 0.1 M glycine.HCl pH 2.5. The influence of elution volume and time on the recovery of active MAB and the overall purification factor were studied. The best conditions enabled the recovery of 50.2% of active MAB with a purification factor of 6.2. A further dialysis against 50 mM Tris.HCl pH 8.0 increased the activity yield and the purification factor to 68.4% and 8.3, respectively. This result showed that part of the antibody activity loss during affinity precipitation was due to a reversible inactivation process, being easily recovered after a refining dialysis step.

Antibodies, Monoclonal↗

PubMed1997

The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation.

The mechanism of 2,2,2-trichloroacetic acid (TCA)-induced precipitation of proteins is studied. The TCA-induced protein precipitation curves are observed to be U-shaped. It is bound that the protein-precipitate-inducing effects of TCA are due to the three chloro groups in the molecule. Using cardiotoxin III (CTX III) isolated from the Taiwan cobra (Naja naja atra), as a model protein, we attempt to understand the molecular basis for the TCA-induced effects. Employing circular dichroism, proton-deuterium exchange in conjunction with conventional 2D NMR techniques, and 1-anilino naphthalene-8-sulfonate-binding experiments, we demonstrate that CTX III is in a partially structured state similar to the 'A state' in 3% w/v TCA. It is postulated that the formation of this 'sticky' partial structured 'A state' in the TCA-induced unfolding pathway is responsible for the acid-induced protein precipitation.

Chemical Precipitation↗

PubMed2003

Simple method of preparation of rat colonocyte apical membranes using polyethylene glycol precipitation.

BACKGROUND: Although a number of methods are available for the preparation of brush border membranes (BBM) from the small intestine, very few studies have been performed on the isolation of colonic apical membranes (CAM). Cation precipitation is one of the methods used for the isolation of these membranes, which can result in altered lipid composition as a result of activation of phospholipase. We have earlier established a method for BBM isolation using polyethylene glycol (PEG) precipitation. In the present study, CAM from isolated colonocytes were prepared using PEG and the isolated membranes were characterized. METHODS: The CAM were prepared from isolated rat colonocytes using PEG precipitation and differential centrifugation. Purity was assessed by enrichment of the marker enzymes and contamination by other subcellular particles. Lipid composition and sugar components were analyzed in the isolated membranes. RESULTS: The CAM showed 11-12-fold enrichment in alkaline phosphatase and 9-10-fold enrichment in gamma-glutamyl transpeptidase as compared with the cell homogenate. The final membrane preparation also showed good vesicle formation as seen by electron microscopy. There was very little contamination of basolateral membranes, microsomes or lysosomes in the final membrane preparation. Analysis of sugars indicated high content of fucose and sialic acid as compared with hexoses. Lipid analysis indicated the presence of various neutral and phospholipids and a cholesterol/phospholipid ratio of 0.65 was seen. CONCLUSION: This study has shown a simple method of CAM preparation using PEG precipitation, which is less time-consuming than other methods and comparatively pure.

Animals↗

PubMedDate not supplied

Evaluation of factor VIII-rich cryoprecipitate and the plasma fibronectin-rich, heparin-precipitable fraction prepared from single-donor plasma units.

A plasma fibronectin-rich component was prepared by heparin-induced 4 degrees C precipitation of fresh or stored (21 days at 4 degrees C), single-donor plasma. The recovery of plasma fibronectin was 45 percent at a concentration of 0.05 mg heparin per ml (7.5 units/ml) and 75 percent at 0.1 mg per ml (15 units/ml). The biologic activity of plasma fibronectin, as assessed by the spreading of Chinese hamster ovary cells or attachment of monocytes to gelatin-coated surfaces, was similar to that of plasma fibronectin concentrates made from fresh or stored plasma. Only 20 to 30 percent of the factor VIII activity in fresh plasma was recovered in cryoprecipitate produced after the heparin-induced precipitate containing fibronectin was removed. Cryoprecipitate prepared from the supernatant plasma that remains after heparin-induced cold precipitation in the presence of CaCl2 (5 mM) contained approximately 50 percent less factor VIII. The relatively low recovery of factor VIII in cryoprecipitate prepared from fibronectin-depleted plasma makes cryoprecipitation an unsuitable method of producing fibronectin-rich and factor VIII-rich components effectively from a single unit of fresh plasma. However, heparin-induced cold precipitation provides an efficient method for preparing plasma fibronectin concentrates from small plasma pools or single units of stored or fresh plasma.

Blood Preservation↗

PubMed1970

Precipitation of proteins by vinblastine and calcium ions.

We have found that vinblastine sulfate can precipitate a number of proteins in addition to microtubule protein. Those proteins which precipitate with vinblastine sulfate, all of which were derived from structure, also precipitate with Ca(2+) ions. Our results suggest that vinblastine sulfate, presumably acting as a cation, precipitates proteins by combining with sites which can also combine with Ca(2+) ions.

Animals↗

PubMed2003

Treatment of metal-contaminated water and vertical distribution of metal precipitates in an upflow anaerobic bioreactor.

A lab-scale upflow anaerobic bioreactor filled with granular sludge and cow manure was operated for 140 days to determine the mechanism of metal removal and the vertical distribution of metal precipitates. Heavy metal ions were removed in the order of Cu2+, Cd2+, Zn2+, Fe2+ and Mn2+ with respect to the height in the reactor. The solid phase analysis showed that the heavy metals were mostly precipitated in the form of metal sulfides by sulfate reduction The contents of metal precipitates in the reactor were as follows: (i) Cd and Zn were highest in the bottom, (ii) Fe was highest at the low-middle layer, and (iii) Mn was increased with the height in the reactor. The vertical distribution of metal sulfides in the reactor was directly related to the solubility product (Ksp). Results obtained in this study suggest a feasibility of the application to separate precipitation metal-containing wastewater.

Bacteria, Anaerobic↗

PubMed2001

Optimalisation of magnesium ammonium phosphate precipitation and its applicability to the removal of ammonium.

Among the physico-chemical abatement technologies, mainly acid scrubbers have been used to control NH3-emission. The disadvantage of this technique is that it yields waste water, highly concentrated in ammonia. In this report, the applicability of the magnesium ammonium phosphate (MAP) process to regenerate the liquid phase, produced by scrubbing NH3-loaded waste gases, was investigated. In the MAP process, ammonium is precipitated as magnesium ammonium phosphate, which can be used as a slow release fertilizer. The influence of a number of parameters, e.g. pH, kinetics, molar ratio NH(+)4/Mg2+/PO(3-)4 on the efficiency of the formation of MAP and on the ammonium removal efficiency was investigated. In this way, optimal conditions were determined for the precipitation reaction. Next to this, interference caused by other precipitation reactions was studied. At aqueous NH(+)4-concentrations of about 600 mg l(-1), ammonium removal efficiencies of 97% could be obtained at a molar ratio NH(+)4/Mg2+/PO(3-)4 of 1/1.5/1.5. To obtain this result, the pH was continuously adjusted to a value of 9 during the reaction. According to this study, it is obvious that the MAP-precipitation technology offers opportunities for ammonium removal from scrubbing liquids. The practical applicability of the MAP-process in waste gas treatment systems, however, should be the subject for further investigations.

Chemical Precipitation↗

PubMed2002

Isolation and biochemical characterization of extracellular polymeric secretions (EPS) from modern soft marine stromatolites (Bahamas) and its inhibitory effect on CaCO3 precipitation.

Bahamian soft marine stromatolites consist of cyanobacterial biofilms and carbonate sand grains (ooids) embedded in their extracellular polymeric secretions (EPS). EPS were isolated from natural marine stromatolites and the laboratory cultured stromatolite forming cyanobacterium isolate Schizothix sp. Laboratory investigations were conducted to examine biochemical characteristics and the role of EPS in the inhibition of CaCO3 precipitation. EPS consisted of acid polysaccharides and proteins. SDS-PAGE and amino acid analysis suggested that EPS from both soft marine stromatolite and Schizothrix sp. mat contained small proteins (38 kD and 45 kD) enriched in aspartic acid and glutamic acid. Also, immuno blotting suggested that natural EPS contain high molecular weight acid polysaccharide (500 k) which may represent cross-linked products of laboratory cultured Schizothrix sp. acid polysaccharide (300 k). EPS from both soft marine stromatolite and laboratory cultured Schizothrix sp. inhibited CaCO3 precipitation in vitro, as determined using pH drift assays examining pH decrease which occur in response to CaCO3 precipitation. PH drift assays of enzymatically and chemically modified EPS isolated from soft marine stromatolite and laboratory cultured Schizothrix sp. indicated that both uronic acids and protein fractions may be involved in the inhibition of CaCO3 precipitation.

Amino Acids↗

PubMed1992

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.

Aggrecans↗

PubMed1977

Isolation of polyoma virus-induced surface antigens in hamster cells: potassium chloride solubilization and differential precipitation.

Surface antigens were extracted in soluble and active form with 3 M KCL from polyoma virus-transformed and embryonic hamster cells. These were tumor-specific transplantation antigens (TSTA), shown by tumor rejection, and a surface (S) antigen, demonstrated by the inhibition of surface fluorescence on living polyoma virus-transformed cells. The extracts were fractionated by salting out with ammonium sulfate. In tumor cell extracts, all TSTA activity and a part of S antigen activity were found in the fraction precipitated with 60% saturation in (NH/)2SO4. Another part of S antigen activity was found in the fraction precipitating at 80% saturation in tumor cell extracts. The precipitate at 60% saturation of embryonic cell extracts also had a part of S antigen activity. Receptor site activity for concanavalin A was also retained after solubilization and was confined to the 40% saturation (NH4)2SO4 precipitate in the case of tumor and embryonic cell extracts.

Animals↗

PubMedDate not supplied

Precipitation of fibrin monomers and fibrin degradation products by ristocetin.

Ristocetin, at relatively low concentrations (1.0 mg/ml-1.5 mg/ml), can selectively precipitate fibrin monomers and fibrin degradation products (fdp) from plasma without effect on fibrinogen or fibrinogen degradation products (FDP). 125I-labeled fibrin monomers and fibrin degradation products were precipitated by ristocetin when their plasma concentrations were greater than 0.25 microgram/ml and 50 microgram/ml, respectively. In order to obtain a visible precipitated, 2 microgram/ml of fibrin monomers of 50 to 100 microgram/ml of fibrin degradation products were necessary. These effects were optimally observed under the following conditions: (1) temperature, 20 C to 37 C; (2) pH, 7.0 to 7.5; and (3) incubation time, 15 to 60 minutes. Late-fibrin degradation products are approximately eight times less sensitive to ristocetin-induced precipitation than early-fibrin degradation products. Plasma medium is essential for the differentiation of fibrin monomers and fibrin degradation products from fibrinogen and fibrinogen degradation products by ristocetin. These results suggest that the specific detection of fibrin monomers and fibrin degradation products in plasma may be easily performed by ristocetin.

Anticoagulants↗

PubMedDate not supplied

Separation of IgG and IgM from albumin in citrated human plasma using electrodialysis and metal ion affinity precipitation.

Plasmapheresis is the process by which plasma is separated from whole blood for therapeutic purposes. The primary reason to perform plasmapheresis is to remove immunoglobulins and not to necessarily remove the remainder of the plasma proteins. Nevertheless, most plasmapheresis is performed by centrifugal separation to remove bulk plasma. We have investigated whether electrodialysis and metal ion affinity precipitation can be used to selectively remove immunoglobulins from citrated plasma and potentially serve as an adjunctive technique to centrifugal plasmapheresis. Using a commercial electrodialysis device, we have desalted citrated plasma to separate immunoglobulins from albumin, and have determined the effects of pH adjustment and addition of zinc acetate for concomitant metal ion affinity precipitation. With desalted pH adjusted citrated plasma containing 1.5 mM zinc acetate, we achieved more than 80% recovery of albumin with removal of almost 60% of the immunoglobulin (Ig)G. Almost 80% of polyclonal IgM and 90% of monoclonal IgM was also removed. IgA was not effectively removed under any of the conditions tested. Selective precipitation with electrodialysis and zinc acetate precipitation appears to be an effective technique for the separation of IgG and IgM from albumin in citrated plasma.

Antibodies, Monoclonal↗

PubMed1989

Selective precipitation of 14 kDa stone/thread proteins by concentration of pancreaticobiliary secretions: relevance to pancreatic ductal obstruction, pancreatic failure, and CF.

Concentrated proteins in pancreatic juice could precipitate and plug pancreatic ducts, initiating pancreatic disease. In cystic fibrosis (CF), pancreatic fluid secretion is impaired due to defective anion transport and proteins are hyperconcentrated. To determine whether proteins in pancreatic secretions precipitate selectively or nonspecifically, duodenal secretions were obtained from subjects with and without cystic fibrosis, during pancreatic stimulation with cholecystokinin (CCK) and secretin, dialyzed against phosphate-buffered saline (PBS), and concentrated in stages by ultrafiltration. Precipitates obtained by centrifugation at 15,600 g for 10 min at 4 degrees C were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The only protein band regularly enriched in precipitates from 10 CF and 9 non-CF samples had an Mr of approximately 14,000. Monoclonal antibodies, raised independently in two laboratories against either pancreatic stone protein (PSP) or pancreatic thread protein (PTP), reacted with the Mr 14,000 protein(s). Differential extraction of PTP and PSP by 0.1N HCl and 10% sodium citrate produced Mr 14,000 products that reacted equally with each monoclonal antibody (MAB). Two-dimensional gel electrophoresis demonstrated two Mr 14,000 spots in each extract with pI's of 5.8 and 6.0. Each spot reacted equivalently with the MABs for PSP and PTP. PSP/PTP type proteins are sparingly soluble in pancreatic secretions and could contribute to protein plugging in pancreatic disease.

Blotting, Western↗

PubMed1982

Polyvinylpyrrolidone (PVP): a new precipitating agent for human and bovine factor VIII and fibrinogen.

The PVP precipitating properties for human and bovine factor VIII and fibrinogen were studied and a new technique for the fractionation of factor VIII and fibrinogen was developed. The precipitation was performed at different temperatures and different PVP concentrations and the best conditions for the technique were chosen. The technique consists of two steps: (a) precipitation of factor VIII from undiluted plasma with PVP and (b) washing of the precipitate with a glycine--saline solution. The final concentrate contains 90% of the factor VIII and 20% of the fibrinogen of the original plasma.

Animals↗

PubMed1981

Measurement of alum precipitated allergen extracts of RAST inhibition methods.

The feasibility of measuring the allergenic activities of alum precipitated pollen extracts by means of the radioallergosorbent test (RAST) was investigated. Different batches of aqueous and alum precipitated birch and mixed grass pollen extracts were used as antigens. The alum precipitates could be readily dissolved with 0.1 M EDTA (pH 9.0) without any loss in allergenicity. No significant differences in RAST activities of undissolved and dissolved alum precipitated allergen extracts could be detected in terms of ability to inhibit the binding of allergen specific IgE to solid-phase allergens.

Allergens↗

PubMed1982

Detection of Aspergillus antigen and anti-Aspergillus precipitating antibodies by a new method: the radial immunoelectro-osmophoresis (RIEOP).

A new method of Immunoprecipitation-in-gel, the Radial Immunoelectro-osmophoresis (RIEOP) has been applied to detect precipitating anti-Aspergillus fumigatus antibodies and to study the antigens present in various antigenic extracts of this fungus. The results obtained with this method have been compared with those obtained when applying the Comparative Double Diffusion (CDD) and the Immunoelectro-osmophoresis (IEOP). The data presented prove that the RIEOP method is more sensitive than CDD and the IEOP in: (1) detecting precipitating anti-Aspergillus antibodies, since (a) precipitating bands are detected with higher serum dilutions, and (b) when using undiluted serum, the number of precipitating bands obtained by RIEOP is higher; and (2) studying antigens present in the antigenic extracts, since (a) the number of antigens detected by RIEOP is higher than that by CDD, and (b) identity/non-identity reactions can be studied among the antigens present in the various extracts.

Antibodies, Fungal↗

PubMed1976

Precipitating antibodies against Micropolyspora phaeni in sera from mushroom workers.

A new method for determining precipitating antibodies in allergic alveolitis is presented. The principle used is immunoelectrophoresis, either as a counterelectrophoresis or as a crossed immunoelectrophoresis with intermediate gel. Twenty-two mushroom workers without anamnestic signs of allergic alveolitis and 15 control persons were investigated. Twelve mushroom workers (55%) had precipitating antibodies as determined by crossed immunoelectrophoresis, and nine of these persons had antibodies demonstrated with counterelectrophoresis. Thus, the sensitivity seems greater in the crossed immunoelectrophoresis, but we find that the simpler counterelectrophoresis is an excellent screening procedure in patients suspected of allergic alveolitis. In the controls only one person had precipitating antibodies, though the antigen tested for Micropolyspora phaeni exists ubiquitously. It seems probable from this study that the antigenic load is important, although it is remarkable that no mushroom worker with precipitating antibodies against M. phaeni had ever experienced symptoms compatible with allergic alveolitis.

Actinomycetales Infections↗