New Approach For Isolation Research Articles

Determination of Glycerophospholipids in Biological Material Using High-Performance Liquid Chromatography with Charged Aerosol Detector HPLC-CAD-A New Approach for Isolation and Quantification.

The method of using high-performance liquid chromatography with a charged aerosol detector method (HPLC-CAD) was developed for the separation and determination of phospholipids isolated from cell membranes. The established cell lines—normal and neoplastic prostate cells and normal skin fibroblasts and melanoma cells—were selected for the study. Chromatographic separation was performed in the diol stationary phase using a gradient elution based on a mixture of n-hexane, isopropanol and water with the addition of triethylamine and acetic acid as buffer additives. Taking the elements of the Folch and Bligh–Dyer methods, an improved procedure for lipid isolation from biological material was devised. Ultrasound-assisted extraction included three extraction steps and changed the composition of the extraction solvent, which led to higher recovery of the tested phospholipids. This method was validated by assessing the analytical range, precision, intermediate precision and accuracy. The analytical range was adjusted to the expected concentrations in cell extracts of various origins (from 40 µg/mL for PS up to 10 mg/mL for PC). Both precision and intermediate precision were at a similar level and ranged from 3.5% to 9.0%. The recovery for all determined phospholipids was found to be between 95% and 110%. The robustness of the method in terms of the use of equivalent columns was also confirmed. Due to the curvilinear response of CAD, the quantification was based on an internal standard method combined with a power function transformation of the normalized peak areas, allowing the linearization of the signal with an R2 greater than 0.996. The developed method was applied for the isolation and determination of glycerophospholipids from cell membranes, showing that the profile of the tested substances was characteristic of various types of cells. This method can be used to assess changes in metabolism between normal cells and neoplastic cells or cells with certain pathologies or genetic changes.

Employing marine invertebrate cell culture media for isolation and cultivation of thraustochytrids

Abstract Thraustochytrids, a common group of marine eukaryotic protists, have drawn considerable scientific and industrial interest due to their ability to synthesize high levels of bioactive compounds, including polyunsaturated fatty acids, docosahexaenoic acid, squalene and carotenoids, and their new applications for biofuels. The pharmaceutical and industrial potential of thraustochytrids necessitate effective isolation of new strains and establishment of axenic cultures. To date, existing isolation protocols have used baiting and direct plating methods to generate axenic cultures with varied media compositions that contain peptone and yeast extracts as nitrogen sources, glucose as carbon source, seawater and antibiotics. Here we reveal a new approach for the isolation of thraustochytrids from tissues of marine invertebrates using (a) primary cell cultures in a liquid medium containing basal medium, 50% artificial seawater, vitamins, proteins and antibiotics, and (b) cultivation in the same cell culture medium. Using the colonial tunicate Botryllus schlosseri as a model system, thraustochytrid cells thrived in the medium from the day of extraction, grew and proliferated for the next five weeks (five-passages, up to 1.9 × 106 cells ml−1 in passage 5; 1.45-fold multiplication week−1). This new approach for isolation and cultivation of axenic thraustochytrid cultures enables the isolation of new species with promising bioactive compounds.

Abstract 5701: Innovative approach for isolation of circulating cell-free DNA from liquid biopsies

Abstract We developed a new approach for isolation of circulating cell-free DNA (cfDNA) from liquid biopsies (plasma or serum). Our technology is based on using the proprietary bi-functional substance X (SubX®) that binds DNA under physiological conditions (e.g. directly in biological liquids), followed by adsorption of [DNA- SubX®] complex on a solid phase matrix. Since SubX®) captures DNA via phosphate groups it allows for elimination of bias related to both AT/CG content and DNA fragments length, thus improving extraction efficacy and accuracy of downstream applications. All currently available commercial kits are [silica-chaotropic salt]-based and exhibit bias for either short or long DNA fragments, as well as for the GC content. In addition, such systems require at least 4- fold dilution of starting volume of bio liquid with concentrated (~5.0 M) guanidine thiocyanate (GTC) to keep resulted GTC concentration at about least 3.0 M. Thus, already low concentrations of cfDNA are significantly reduced and may negatively influence extraction efficacy of low abundance DNA fragments. Since our method does not require chaotropic salt for DNA capture, no dilution of starting material (and thus cfDNA) takes place. DNA-binding matrix SubX® allows either scaling the procedure to either large volumes of starting material or extra low volumes and adaptation of the protocol for 96/384 well format. Separation of cfDNA from the bulk of proteins occurs in a single vortex-spin step without employing chaotropic agents thus speeding-up isolation procedure and reducing the costs. The whole procedure can be completed within an hour and cfDNA is eluted in small volume of TE buffer. Average DNA yield from 0.2 ml of double centrifuged blood plasma from normal donors, cancer and multiple sclerosis patients was 7.5±4.2 ng (n=96). Citation Format: Andrei Malykh, Vladimir Evtushenko. Innovative approach for isolation of circulating cell-free DNA from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5701. doi:10.1158/1538-7445.AM2017-5701

New Approach for Isolation of Individual Caseins from Cow Milk by the Preparative Electrophoresis

This paper carried out a comparative analysis of different types of electrophoretic systems which were used for the analysis of casein complex from cow milk (polyacrylamide gel electrophoresis: for the neutral and acidic native conditions, in gradient variant, the presence of sodium dodecyl sulphate or with including urea). Taking into attention the separation efficiency, complexity of electrophoretic system, the impact of system components, we have selected the anode system of the homogeneous gel in the presence of urea as the basis for the preparation of casein fractions. It also changed the composition and the structure of the electrophoretic apparatus. The changes allow purification of casein fractions up to several grams during one stage of treatment (for 5 hours). The purified casein fractions were tested for the homogeneity and have been recommended for using in the biomedical researches, including the processes of the formation of the bioactive peptides.

Thiolsulfonate functionalized polystyrene resin: Preparation and application in the isolation and identification of electrophilic mutagens

A new approach for isolation and identification of elecrtophilic mutagens from complex matrix was developed. Thiosulfonic anion was immobilized onto polystyrene beads and used as separation media. Potassium polystyryl-thiosulfonate, prepared from polystyrylsulfonyl chloride and KHS, was observed to selectively react with model electrophilic mutagens such as alkyl halides, α-chloroketones and α-chloroesters to produce polystyryl-thiosulfonic esters. After separation from other nonreactive organic compounds, the beads then reacted with ethanethiol to produce unsymmetrical ethyl disulfides which are easily detected by GC/MS. For one mutagenic compound, only one unsymmetrical disulfide was found to contain its structure part. Thus, the structure of the parent mutagens could be deduced from that of the unsymmetrical disulfides. The degree of functionalization of the potassium polystyryl-thiosulfonate resin was 1.11 mmol/g. Its reactivity was discussed and its recycling method was reported here.

Purification and characterization of the Ca2+/calmodulin-dependent protein kinase II from chicken forebrain.

CaM kinase II is known to be enriched in mammalian and avian brains. To determine the holoenzymic composition and functional characteristics of this kinase, a new approach for isolation was applied to isolate it from the chicken forebrain. Forebrains of hatched 45-d chicken were dissected, homogenized, and centrifuged. The supernatant was loaded onto a CaM-agarose affinity column and the calmodulin-binding proteins were eluted with EGTA. Selected eluates were loaded onto the antibody-agarose affinity column, which was prepared with monoclonal antibody (MAb) (6G9) to the CaM kinase II alpha subunit. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and either silver-stained or blotted onto a nitrocellulose membrane. The protein composition and the immunoreactivity of the antibody-agarose affinity eluate fractions were analyzed with a densitometric scanner. Silver staining of gels showed that the beta subunit doublet, the beta' subunit, and a putative substrate were coeluted with the alpha subunit from the antibody affinity column although only the alpha subunit bound the 6G9 antibody. Scintillation counting showed that the autophosphorylation of the kinase was significantly reduced in the eluate from the antibody affinity column. Whereas silver staining indicated an increase in the relative amount of alpha subunit had occurred during purification, phosphorylation assays indicated an increase in the relative amount of the alpha subunit after the last purification step. A possible reason for this is discussed. The presence of beta/beta' subunits in the antibody-agarose affinity eluate indicated the existence of an alpha beta/beta' heteropolymer. The phosphorylation assay was not a good indication of the amount of purification because of the loss of enzyme activity following purification. In contrast, the immunoassay indicated a 97-fold purification from the cytosolic fraction was achieved using the method. In conclusion, the data indicate the existence of the CaM kinase II alpha beta/beta' heteropolymer in the chicken forebrain.

Directed integration of an F′ plasmid by integrative suppression

A new approach for isolation of a plaque forming lambda specialized transducing phage is described. It consists of directed transposition of an F' plasmid into the gal region of a dnaAts galE- Escherichia coli strain by integrative suppression and deletion of the chlD region in order to shorten the distance between the marker of interest on the F' and the prophage serving to prepare an LFT1 lysate. An F' danC+thr+ plasmid was used here and lambdadthr and lambdaddnaC phages were isolated. In addition, lambdapdnaC was obtained from a double lysogen for lambdaddnaC and lambda b2.