Comparative study of three novel ion exchange resins with DEAE-cellulose for the purification of Trypanosoma evansi.

Ion exchange chromatography is a method that uses the different surface charges of trypanosomes and blood cells to separate them. This makes it possible to use molecular and immunological methods to diagnose or study these protozoans. DEAE-cellulose resin is commonly used to perform this method. The goal of this study was to compare three novel chromatographic resins designated as PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®). The resins were evaluated based on their ability to isolate the parasite, purification time, examination of parasite viability and morphology, and trypanosome recovery potential after passing through the columns. In terms of the evaluated parameters, there was no significant difference between DEAE-cellulose and the three tested resins in most experiments. However, PURIFICA™ (Y-C2N®, Y-HONOH®, and Y-CNC3®) resins are less expensive and easier to prepare than DEAE-Cellulose, making them an alternative for the purification of Trypanosoma evansi.

Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus.

BACKGROUND: In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. RESULT: The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of ß-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. CONCLUSION: The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas ß-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste.

Isolation and Characterization of a Phaseolus vulgaris Trypsin Inhibitor with Antiproliferative Activity on Leukemia and Lymphoma Cells.

A 17.5-kDa trypsin inhibitor was purified from Phaseolus vulgaris cv. "gold bean" with an isolation protocol including ion exchange chromatography on DEAE-cellulose (Diethylaminoethyl-cellulose), affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-sepharose (Sulfopropyl-sepharose), and gel filtration by FPLC (Fast protein liquid chromatography) on Superdex 75. It dose-dependently inhibited trypsin with an IC50 value of 0.4 µM, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. It inhibited [methyl-³H] thymidine incorporation by leukemia L1210 cells and lymphoma MBL2 cells with an IC50 value of 2.3 µM and 2.5 µM, respectively. The inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to 100 µM.

Aqueous two-phase systems: A simple methodology to obtain mixtures enriched in main toxins of Bothrops alternatus venom.

Phospholipase A2 (PLA2) and protease (P) are enzymes responsible of myotoxic, edematogenic and hemostasis disorder effects observed in the envenomation by Bothrops alternatus pitviper. Their partitioning coefficient (Kp) in different polyethyleneglycol/potassium phosphate aqueous two-phase systems (ATPSs) was determined in order to both achieve a better understanding of the partitioning mechanism and define optimal conditions for toxin isolation. Polyethyleneglycols (PEGs) of molecular weights 1000; 3350; 6000 and 8000; different temperatures (5, 20 and 37 °C) and phase volume ratios of 0.5; 1 and 2 were assayed. PLA2 partitioned preferentially to the top phase while P mainly distributed to the bottom phase. Either entropically- or enthalpically-driven mechanisms were involved in each case (PLA2 and P). The aqueous two-phase system formed by PEG of MW 3350 (12.20% wt/wt) and KPi pH 7.0 (11.82% wt/wt) with a volume ratio of one and a load of 1.25 mg of venom/g of system showed to be the most efficient to recover both enzymes. It allowed obtaining the 72% of PLA2 in the top phase with a purification factor of 2 and the 82% of P at the bottom phase simultaneously. A further adsorption batch step with DEAE-cellulose was used to remove satisfactorily the PEG from the top phase and recover the active PLA2. The proposed methodology is simple, inexpensive, and only requires professionals trained in handling basic laboratory equipment. It could be easily adoptable by developing countries in which the snakebite accidents cause considerable morbidity and mortality.

SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096: Robust stability and unusual non-cumbersome purification.

Present study underlines an unusual non-cumbersome-powerful strategy for purification of SHG10 keratinolytic alkaline protease from Bacillus licheniformis SHG10 DSM 28096 with robust stability properties. The enzyme was impressively purified to homogeneity with specific activity, purification fold, and yield of 613.82 U mg-1 , 58.91 and 99%, respectively, via a sequential two-step purification strategy: precipitation with 65% (NH4 )2 SO4 and flow through fractions of DEAE-cellulose DE 53 column. SDS-PAGE conferred a monomeric enzyme with a molecular mass of 30.4 kDa. The enzyme demonstrated optimal activity at pH (10.0-11.0) and at 65 °C. It exhibited full stability at pH (6.0-11.0) over 38 h at 4 °C and at 65 °C for 15 min. Remarkable enhanced enzyme activity (130.15 and 126.37%) was retained in presence of commercial laundry detergents Oxi and Ariel after 1 h, respectively. Organic solvent stability of the enzyme was verified in butanol, ether, acetonitrile, isopropanol, and chloroform. Imposingly, full storage stability (100%) of the enzyme along 1 year in -20 °C was confirmed. Km -Vmax was 0.00174 mM-534.2 mM Sub · min-1 · mg protein-1 and 1.266 mg-28.89 mg Sub · h-1 · mg protein-1 on N-Suc-Ala-Ala-Pro-Phe-pNA and keratin azure, respectively. Robust stability properties of SHG10 keratinolytic alkaline protease along with rapid-efficient purification underpin its potential commercialization for industrial exploitation.

Purification and characterization of alginate lyase from locally isolated marine Pseudomonas stutzeri MSEA04.

An alginate lyase with high specific enzyme activity was purified from Pseudomonas stutzeri MSEA04, isolated from marine brown algae. The alginate lyase was purified by precipitation with ammonium sulphate, acetone and ethanol individually. 70% ethanol fraction showed maximum specific activity (133.3 U/mg). This fraction was re-purified by anion exchange chromatography DEAE- Cellulose A-52. The loaded protein was separated into 3 peaks. The second protein peak was the major one which contained 48.2% of the total protein recovered and 79.4% of the total recovered activity. The collected fractions of this peak were subjected to further purification by re-chromatography on Sephadex G-100. Alginate lyase activity was fractionated in the Sephadex column into one major peak, and the specific activity of this fraction reached 116 U/mg. The optimal substrate concentration, pH and temperature for alginate lyase activity were 8 mg/ml, pH 7.5 and 37 °C, respectively. While, Km and Vmax values were 1.07 mg alginate/ ml and 128.2 U/mg protein, respectively. The enzyme was partially stable below 50 °C, and the activity of the enzyme was strongly enhanced by K(+), and strongly inhibited by Ba(+2), Cd(+2), Fe(+2) and Zn(+2). The purified enzyme yielded a single band on SDS-PAGE with molecular weight (40.0 kDa).

A novel ribonuclease with HIV-1 reverse transcriptase inhibitory activity purified from the fungus Ramaria formosa.

A purification protocol that encompassed anion exchange chromatography (EC) on DEAE-cellulose, cation EC on CM-cellulose, anion EC on Q-Sepharose, and fast protein liquid chromatography-gel filtration of Superdex 75 was used to isolate a ribonuclease from dried fruiting bodies of Ramaria formosa. The ribonuclease was unadsorbed on CM-cellulose but adsorbed on both DEAE-cellulose and Q-Sepharose. It displayed a molecular mass of 29-kDa in both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ranking of its ribonucleolytic activity toward polyhomoribonucleotides was poly(U) > poly(C) > poly(G) > poly(A). It exhibited a pH optimum of pH 5 and a temperature optimum at 60 °C. The ribonuclease inhibited HIV-1 reverse transcriptase with an IC50 of 3 µM.

Ni speciation in tea infusions by monolithic chromatography--ICP-MS and Q-TOF-MS.

For humans, Ni is not considered to be an essential trace element. Its compounds, at levels present in foodstuffs and drinks, are generally considered to be safe for consumption, but for individuals who already suffer from contact allergy to Ni and may be subject to develop systemic reactions from its dietary ingestion, dietary exposure to Ni must be kept under control. Being the second most popular beverage, tea is a potential source of dietary Ni. Present knowledge on its speciation in tea infusions is poor. Therefore, complete speciation analysis, consisting of separation by liquid chromatography using a weak CIM DEAE-1 monolithic column, "on-line" detection by inductively coupled plasma mass spectrometry (ICP-MS) and "off-line" identification of ligands by hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MS), was implemented for the first time to study Ni speciation in tea infusions. Total concentrations of Ni in dry leaves of white, green, oolong and black tea (Camellia sinensis) and flowers of herbal chamomile (Matricaria chamomilla) and hibiscus (Hibiscus sabdariffa) tea were determined after microwave digestion by ICP-MS. They lay between 1.21 and 14.4 mg kg(-1). Good agreement between the determined and the certified values of the Ni content in the standard reference material SRM 1573a tomato leaves confirmed the accuracy of the total Ni determination. During the infusion process, up to 85 % of Ni was extracted from tea leaves or flowers. Separation of Ni species was completed in 10 min by applying aqueous linear gradient elution with 0.6 mol L(-1) NH(4)NO(3). Ni was found to be present in the chromatographic fraction in which quinic acid was identified by Q-TOF in all the tea infusions analysed, which had pH values between 5.6 and 6.0. The only exception was the infusion of hibiscus tea with a pH of 2.7, where results of speciation analysis showed that Ni is present in its divalent ionic form.

Properties of cell wall polysaccharides of raw nectarine fruits after treatment under conditions that modulate gastric digestion.

The results of the study of the physicochemical properties of the high-molecular-weight soluble and insoluble components of nectarine cell walls obtained by fruit treatment under conditions that modulate of gastric digestion are presented. Homogenized nectarine fruits were sequentially treated by natural saliva and simulated gastric fluid (SGF) at pH 1.8 and 3.0. The isolated polysaccharides were compared with polysaccharides obtained by sequential extraction of nectarine fruit with cold, hot, and acidified water, solutions of ammonium oxalate and sodium carbonate. As a result, high-molecular-weight water-soluble pectic polysaccharides, weakly bound in the cell wall, were dissolved in the simulated gastric fluid, regardless of pH. Homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) were identified in all pectins. It was shown that their quantity and ability to form highly viscous solutions determine high values of the rheological characteristics of the nectarine mixture formed under simulated gastric conditions. The modifications occurring with the insoluble components under the influence of acidity of SGF were importance. They determined difference in the physicochemical properties of both the insoluble fibres and the nectarine mixtures.

Production of cold-active pectinases by three novel Cladosporium species isolated from Egypt and application of the most active enzyme.

Cladosporium parasphaerospermum, Cladosporium chlamydosporigenum, and Cladosporium compactisporum have all been discovered and characterized as new Cladosporium species. The three new species seemed to generate cold-active pectinases with high activity at pH 6.0 and 10 °C, pH 6.0 and 15 °C, and pH 5.0 and 15 °C, respectively, with the most active being C. parasphaerospermum pectinase. In submerged fermentation (SmF), C. parasphaerospermum produced the most cold-active pectinase with the highest activity and specific activity (28.84 U/mL and 3797 U/mg) after 8 days. C. parasphaerospermum cold-active pectinase was isolated using DEAE-Cellulose anion exchange resin and a Sephadex G 100 gel filtration column. The enzyme was purified 214.4-fold and 406.4-fold greater than the fermentation medium using DEAE-cellulose and Sephadex G 100, respectively. At pH 7.0 and 10 °C, pure pectinase had the highest activity (6684 U/mg), with Km and Vmax determined to be 26.625 mg/mL and 312.5 U/min, respectively. At 5 mM/mL, EDTA, MgCl2, and SDS inhibited the activity of pure pectinase by 99.21, 96.03, and 94.45%, respectively. The addition of 10 U/mL pure pectinase enhanced the yield of apple, orange, apricot, and peach juice by 17, 20, 13, and 24%, respectively, and improved the clarity and colour of orange juice by 194 and 339%, respectively. We can now add cold-active pectinase production to the long list of Cladosporium species that have been identified. We also report three new species that can be used in biotechnological solutions as active microbial pectinase producers. Although further research is needed, these distinct species might be used to decompose difficult and resistant pertinacious wastes as well as clear fruit juices.

Polysaccharides from Hericium erinaceus Fruiting Bodies: Structural Characterization, Immunomodulatory Activity and Mechanism.

Five fractions from crude Hericium erinaceus polysaccharides (HEPs), including HEP-1, HEP-2, HEP-3, HEP-4 and HEP-5, were obtained through column chromatography with a DEAE Cellulose-52 column and Sephadex G-100 column. The contents of total carbohydrates and uronic acid in HEPs were 53.36% and 32.56%, respectively. HEPs were mainly composed of Fuc, Gal and Glu in a molar ratio of 7.9:68.4:23.7. Its chemical structure was characterized by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. HEP-1 contains the backbone composed of (1→6)-linked-galactose with branches attached to O-2 of some glucose. The immunological activity assay indicated that HEP-1 significantly promoted the production of nitric oxide, interleukin-6, interleukin-10, interferon-γ and tumor necrosis factor-α and the phosphorylation of signaling molecules. Collectively, these results suggested that HEP-1 could improve immunity via NF-κB, MAPK and PI3K/Akt pathways. Hericium erinaceus polysaccharides might be explored as an immunomodulatory agent for use in dietary supplements.

Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification.

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Biofabrication of skin tissue constructs using alginate, gelatin and diethylaminoethyl cellulose bioink.

INTRODUCTION: Biofabrication of skin tissue equivalents using 3D bioprinting technology has gained much attention in recent times due to the simplicity, the versatility of the technology and its ability in bioengineering biomimetic tissue histology. The key component being the bioink, several groups are actively working on the development of various bioink formulations for optimal skin tissue construction. METHODS: Here, we present alginate (ALG), gelatin (GEL) and diethylaminoethyl cellulose (DCEL) based bioink formulation and its application in bioprinting and biofabrication of skin tissue equivalents. Briefly, DEAE cellulose powder was dispersed in alginate solution with constant stirring at 60 °C to obtain a uniform distribution of cellulose fibers; this was then mixed with GEL solution to prepare the bioink. The formulation was systematically characterized for its morphological, physical, chemical, rheological, biodegradation and biocompatibility properties. The printability, shape fidelity and cell-laden printing were assessed using the CellInk bioprinter. RESULTS: The bioink proved to be a good printable, non-cytotoxic and stable hydrogel formulation. The primary human fibroblast and keratinocyte-loaded 3D bioprinted constructs showed excellent cell viability, collagen synthesis, skin-specific marker and biomimetic tissue histology. CONCLUSION: The results demonstrated the successful formulation of ALG-GEL-DCEL bioink and its application in the development of human skin tissue equivalents with distinct epidermal-dermal histological features.

Characterisation of a haemagglutinin from Hokkaido red bean (Phaseolus vulgaris cv. Hokkaido red bean).

BACKGROUND: A haemagglutinin was purified from Japanese Hokkaido red beans (Phaseolus vulgaris cv. Hokkaido red bean) with a procedure that included three chromatographic media. RESULTS: Haemagglutinating activity was adsorbed on DEAE cellulose, Affi-gel blue gel and Mono S. The pure haemagglutinin was a homodimer and each subunit was around 30 kDa in molecular mass. The haemagglutinating activity of this agglutinin could not be inhibited by a variety of simple sugars at 200 mmol L(-1) concentration including alpha-L-fucose, D(+)-galactose, D(+)-glucose, D(+)-glucosamine, D(-)galactosamine, galacturonic acid, (+)-lactose, D(+)-melibose, L(-)-mannose, D(+)-mannose, D-mannosamine, D(+)-raffinose, L-rhamnose, (+)-xylose and galacturonic acid. The haemagglutinating activity was fully retained at pH 4-11 and at 0-80 degrees C, but was completely lost at extreme pH values (0-2 and 13-14) and at very high temperatures (90 degrees C and 100 degrees C). The haemagglutinin exhibited a weak mitogenic activity toward mouse splenocytes, a stronger anti-proliferative activity than Con A toward HepG2 (human hepatoma) cells and inhibited >80% of HIV-1 reverse transcriptase inhibitory activity at 3.3 micromol L(-1). It was devoid of anti-fungal activity. CONCLUSION: Hokkaido red bean haemagglutinin possesses a potent anti-proliferative effect on HepG2 cells.

Stabilization of Glycosylated ß-Glucosidase by Intramolecular Crosslinking Between Oxidized Glycosidic Chains and Lysine Residues.

Many industrial enzymes can be highly glycosylated, including the ß-glucosidase enzymes. Although glycosylation plays an important role in many biological processes, such chains can cause problems in the multipoint immobilization techniques of the enzymes, since the glycosylated chains can cover the reactive groups of the protein (e.g., Lys) and do not allow those groups to react with reactive groups of the support (e.g., aldehyde and epoxy groups). Nevertheless, the activated glycosylated chains can be used as excellent crosslinking agents. The glycosylated chains when oxidized with periodate can generate aldehyde groups capable of reacting with the amino groups of the protein itself. Such intramolecular crosslinks may have significant stabilizing effects. In this study, we investigated if the intramolecular crosslinking occurs in the oxidized ß-glucosidase and its effect on the stability of the enzyme. For this, the oxidation of glycosidic chains of ß-glucosidase was carried out, allowing to demonstrate the formation of aldehyde groups and subsequent interaction with the amine groups and to verify the stability of the different forms of free enzyme (glycosylated and oxidized). Furthermore, we verified the influence of the glycosidic chains on the immobilization of ß-glucosidase from Aspergillus niger and on the consequent stabilization. The results suggest that intramolecular crosslinking occurred and consequently the oxidized enzyme showed a much greater stabilization than the native enzyme (glycosylated). When the multipoint immobilization was performed in amino-epoxy-agarose supports, the stabilization of the oxidized enzyme increases by a 6-fold factor. The overall stabilization strategy was capable to promote an enzyme stabilization of 120-fold regarding to the soluble unmodified enzyme.

Structure analysis of polysaccharides purified from Cyclocarya paliurus with DEAE-Cellulose and its antioxidant activity in RAW264.7 cells.

CPP was isolated from Cyclocarya paliurus (C. paliurus) and CPP-D was purified from CPP with a further step by DEAE-Cellulose. In this study, the structure and antioxidant activities of these two polysaccharides were investigated. The molecular weight of CPP was determined as 1.15 × 105 Da and the monosaccharides of it were Rha, Ara, Xyl, Man, Glc, Gal in a molar ratio of 0.021:0.237:0.020:0.036:0.454:0.231, while the molecular weight of CPP-D was 9.1 × 103 Da and the monosaccharides of it were Man, Glc, Gal in a molar ratio of 0.235:0.677:0.088. CPP-D consisted of three structural residues →4)-ß-D-Glc-(1→, →2,6)-ß-D-Man-(1→ and →4)-ß-D-Gal. These structures were characterized by SEM, FT-IR, GC-MS, HPGPC, and NMR. The antioxidant assay in RAW264.7 cell showed that both CPP and CPP-D promoted cell viability and antioxidant activity, which decreased the content of MDA and increased the activity of SOD, T-AOC, CAT (P < .05). As a result, CPP-D isolated by DEAE-Cellulose didn't reduce the antioxidant activity of C. paliurus polysaccharide and could enhance the cell viability.

Chemical structure and inhibition on α-glucosidase of polysaccharide with alkaline-extracted from glycyrrhiza inflata residue.

A new neutral polysaccharide, named AGP, was extracted from glycyrrhiza residue by 5% NaOH alkaline solution and purified by DEAE-celluloseand Sephadex G-150. A single and symmetrical peak was shown by HPLC, indicating that AGP is a homogeneous polysaccharide with a molecular weight of 2.89 × 103 KDa. Thespecific rotation of AGP was detected by a polarimeter and it was +45°. Monosaccharide composition analysis indicated that AGP was consisted of l-rhamnose: l-arabinose: d-xylose: d-mannose: d-glucose and d-galactose with a molar ratio of 1:2.33:2.85:0.69:3.05:1.54. The structure of AGP was analyzed by GC-MS, periodate oxidation, Smith degradation, FT-IR, methylation and NMR, which indicated that the AGP was composed of → 6)-ß-d-Glcp-( â†’ backbone and the â†’4)-α-d-Xylp-(1→, →5)-α-l-Araf-(1→, →3)-α-l-Rhap-(1→, →6)-α-d-Galp-(1→, →3,6)-α-Manp-(1→ and →1)-ß-d-Glcp as branches. The results of Congo red experiment and circular dichroism (CD) showed that there was triple helix conformation in AGP. The micro-structure of AGP were detected by scanning electron microscopy (SEM), which concluded that the shape of AGP was a "thin slice" and its structure is not regular. The crystal configuration was identified by X-ray diffraction (XRD), showing that there is no crystal structure. Furthermore, the AGP exhibited certain inhibition activity on α-glucosidase.

Structural characterization and induced copper stress resistance in rice of exopolysaccharides from Lactobacillus plantarum LPC-1.

Excessive accumulation of copper could decrease growth and quality of crops, and little information was currently available on the role of exopolysaccharides (EPS) from Lactobacillus plantarum in inducing copper stress resistance in plants. The main objective of this work was to purify and characterize the EPS produced by our isolated L. plantarum LPC-1, and evaluate its potential protection for rice against copper stress. Firstly, two fractions (EPS-1 and EPS-2) were separated and purified from L. plantarum LPC-1 by DEAE-52 cellulose and Sephadex G-100 cellulose column chromatography. According to the further scanning electron microscope (SEM), ultraviolet-visible (UV), fourier-transform infrared spectroscopy (FTIR) spectroscopy and gas chromatography (GC) analyses, it was observed that EPS-1 and EPS-2 were heteropolysaccharides that were composed of mannose and glucose with molar ratio of 2.40:15.01 and 3.02:11.63, respectively. Additionally, the two fractions possessed considerable antioxidant activities, and EPS-1 had a stronger antioxidant activity than EPS-2 in vitro. Furthermore, exogenous addition of EPS significantly alleviated the toxic effects by copper on rice seedlings. In conclusion, this study provided evidence of the EPS-mediated reduction of copper toxicity in rice seedlings at physiological and biochemical levels, suggesting that EPS could be considered as novel and effective plant immune inducers in crops.

Decontamination of polymerase chain reaction reagents using DEAE-cellulose.

Polymerase chain reaction (PCR) is widely used to detect specific DNA sequences for purposes of microbial identification, clinical diagnosis, and basic research. The most pernicious problem plaguing this technique is contamination of PCR reagents with previously amplified material. We propose a useful tool for PCR reagent purification from contaminating nucleic acid using DEAE-cellulose and present the analysis of this technique for both decontamination efficiency and an effect on the reagent activity. We also show the suitability of the proposed approach for decontamination of the Taq polymerase, monoclonal antibodies to Taq polymerase, and Moloney murine leukemia virus (M-MLV) reverse transcriptase.

Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis.

In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation - whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated napthoylated DEAE (BND)-cellulose in high salt conCentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA and are thus suitable for analysis on 2-D gels.