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PubMed2025

General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.

Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.

Cryoelectron Microscopy↗
PubMed2005

Sample preparation and gas chromatography of primary fatty acid amides.

A method for the isolation of bio-active primary fatty acid amides (PFAM's) from total lipid extract by solid-phase extraction (SPE) was developed and validated. The lowest mass of amide to be loaded and recovered by this method was detected as 0.5 microg using 500 mg of normal phase adsorbent. The isolated PFAM's were separated and quantified by GC/MS and percent recoveries were calculated. An HP-5MS column was able to provide base line separation between the saturated and unsaturated PFAM's whereas clear resolution between geometric and positional isomers having the same number of carbons was obtained using a BPX70 column. The separated amides were all 18 carbon analogs of cis-9-octadecenoamide (oleamide). Detection limits in the single ion monitoring mode were found to be on the order of 10 pg in a 1 microl injection. Solid phase extraction of amides from total lipid extract before GC/MS analysis provides clean detection and interference free analysis.

Amides↗
PubMed2006

Procedures of trophic chain samples preparation for determination of triazines by HPLC and metals by ICP-AES methods.

The aim of this research was monitoring the distribution of atrazine and simazine as well as metals Pb, Cd, Zn, Al, Co, Ni, and V along with trophic chains: soil-vegetables and soil, carrot or grass and meat. Different techniques of herbicides extraction by means of many solvents were examined. Triazines were analysed by means of HPLC, metals by means of ICP-AES. Detection limits: LOD=0.2 microg ml(-1), determination limits: LOQ=0.73 microg ml(-1) for atrazine and LOD=0.3 microg ml(-1), LOQ=1.12 microg ml(-1) for simazine were obtained. The content (microg g(-1)) of simazine in soil was in range: 3.45-8.60, in vegetable roots: 6.62-38.15, in vegetable leaves: 2.45-31.71, in rabbit fat: 0.13-49.90. The content (microg g(-1)) of atrazine in soils was in range: 11.9-13.03, in vegetable roots: 13.61-92.90. In analysed material the particular metals after microwave or dry digestion were determined in range (microg g(-1)): Pb: 6.48-43.18; Cd: 0.11-0.57; Zn: 8.79-51.90; Al: 10.22-24.48; Co: 0.18-3.89; Ni: 0.37-6.36; V: 0.29-1.48.

Aluminum↗
PubMed2005

Increase of the LC-MS/MS sensitivity and detection limits using on-line sample preparation with large volume plasma injection.

Large volume injection (LVI) has systematically been studied to improve LC-MS/MS sensitivity (signal-to-noise ratio, or S/N) and detection limits. The method of LVI was combined with on-line solid phase extraction (on-line SPE) and LC-MS/MS detection for analysis of compounds directly in plasma. It was demonstrated that LVI of plasma with on-line SPE-LC-MS/MS allows for improvement of sensitivity and detection limits without compromising chromatographic peak shape and resolution and inducing significant matrix and signal suppression effects. Furthermore, sensitivity and detection limits improve linearly with the injection volume up to 100 microL. Quantification of the model compounds in plasma demonstrated comparable calibration curve statistics, precision and accuracy for 5, 50 and 100 microL plasma injections.

Animals↗
PubMed2005

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS.

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2000

Optimisation of procedures for the extraction of structural analogues of propranolol with molecular imprinted polymers for sample preparation.

A propranolol-derived molecular imprinted polymer (MIP) was prepared using methacrylic acid as monomer and ethylene glycol dimethacrylate as cross-linker. The extraction properties of five compounds structurally related to propranolol were assessed on the MIP and on a blank polymer made under the same conditions but in the absence of an imprint molecule. Using application from aqueous solution with methanol-water-triethylamine (TEA)-based solvents for elution (i.e. reversed-phase conditions) the MIP showed only marginal selectivity for the compounds on the MIP compared to the blank. Despite the limited selectivity there did appear to be a relationship between structure of the compound (relative to propranolol) and the extent of selective retention. Application of the compounds in toluene with elution using toluene-TEA or toluene-trifluoroacetic acid resulted in the MIP showing dramatically enhanced retention and selectivity of the compounds on the MIP compared to the blank. The enhanced selectivity for extraction on to the MIP relative to the blank, for all compounds using normal-phase solvents seem to be a class effect as there was no apparent relationship between compound structure and retention.

Adrenergic beta-Antagonists↗
PubMed2001

Effects of the matrix and sample preparation on the determination of fluoroquinolone residues in animal tissues.

The effects of aqueous-organic solvent extraction and enzymic digestion sonication procedure on the isolation of spiked fluoroquinolones from poultry tissue have been studied. The highest recovery from spiked tissue was obtained using a mixture of trichloroacetic acid-acetonitrile (8:2) as extractant and an SDB1 cartridge for clean-up purposes. Validation data are presented for enrofloxacin, ciprofloxacin, sarafloxacin and difloxacin. The spiking procedure (spike contact time, spike solvent and matrix) had a small influence on the recovery of fluroquinolones from poultry muscle or liver. The effects of a different extraction on the determination of incurred enrofloxacin and its metabolite, ciprofloxacin, residues in poultry tissues have been investigated. The extraction procedures investigated--aqueous-organic solvent extraction, enzymic digestion or sonication--all gave similar results for incurred fluoroquinolone concentration in poultry muscle after correlation for spike recovery. The highest results were obtained in poultry liver when enzymic digestion has been used.

Animals↗
PubMed1990

Improved sample preparation method for high-performance liquid chromatography of deoxyribonucleoside triphosphates from cell culture extracts.

The accurate determination of deoxyribonucleoside triphosphates in cells is difficult owing to the high concentrations of interfering ribonucleoside triphosphates. The latter can be degraded to their respective bases by periodate oxidation of cell extracts. However, the large amount of bases so produced can interfere with subsequent high-performance liquid chromatographic (HPLC) analysis. The use of a weak ion-exchange cartridge to partially purify and concentrate deoxyribonucleoside triphosphates in periodate-treated cell extracts, prior to HPLC, thus allowing accurate determination is described. The recovery of the deoxyribonucleoside triphosphates is greater than 95%, and greater than 90% of the interfering bases are removed.

Anion Exchange Resins↗
PubMed2002

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization.

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.

Caprolactam↗