Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “PIPET”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources
PubMed2006

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma.

BACKGROUND: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. METHODS: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. RESULTS: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. CONCLUSION: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Antiviral Agents↗
PubMed2006

Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry.

BACKGROUND: Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is an efficient technology for routine determination of immunosuppressants in whole blood; however, time-consuming manual sample preparation remains a significant limitation of this technique. METHODS: Using a commercially available robotic pipetting system (Tecan Freedom EVO), we developed an automated sample-preparation protocol for quantification of tacrolimus in whole blood by LC-MS/MS. Barcode reading, sample resuspension, transfer of whole blood aliquots into a deep-well plate, addition of internal standard solution, mixing, and protein precipitation by addition of an organic solvent is performed by the robotic system. After centrifugation of the plate, the deproteinized supernatants are submitted to on-line solid phase extraction, using column switching prior to LC-MS/MS analysis. The only manual actions within the entire process are decapping of the tubes, and transfer of the deep-well plate from the robotic system to a centrifuge and finally to the HPLC autosampler. Whole blood pools were used to assess the reproducibility of the entire analytical system for measuring tacrolimus concentrations. RESULTS: A total coefficient of variation of 1.7% was found for the entire automated analytical process (n=40; mean tacrolimus concentration, 5.3 microg/L). Close agreement between tacrolimus results obtained after manual and automated sample preparation was observed. CONCLUSIONS: The analytical system described here, comprising automated protein precipitation, on-line solid phase extraction and LC-MS/MS analysis, is convenient and precise, and minimizes hands-on time and the risk of mistakes in the quantification of whole blood immunosuppressant concentrations compared to conventional methods.

Autoanalysis↗
PubMed2006

QPatch: the past, present and future of automated patch clamp.

The QPatch 16 significantly increases throughput for gigaseal patch clamp experiments, making direct measurements in ion channel drug discovery and safety testing feasible. Released to the market in the Autumn of 2004 by Sophion Bioscience, the QPatch originated from work done at NeuroSearch (Denmark) in the early days of automated patch clamp. Today, the QPatch provides many unique features. For example, only the QPatch includes an automated cell preparation station making several hours of unattended operation possible. The 16-channel electrode array, called the QPlate, includes glass-coated microfluidic channels for less compound absorption and, hence, more accurate IC(50) values. The microfluidic pathways also allow for very small amounts of compound used for each experiment ( approximately 5 microl per addition). Only the QPatch has four independent pipetting heads for more efficient liquid handling (especially for ligand-gated ion channel experiments). Patch clamp recordings with the QPatch match the high quality of conventional patch clamp and in some cases the results are even better. For example, only the QPatch includes 100% series resistance compensation for the elimination of false positives due to voltage errors. Finally, the modular QPatch 16 was designed with more channels in mind. The upgrade pathway to 48-channels (the QPatch HT) will be discussed.

Animals↗
PubMed2004

Controlled pseudopod extension of human neutrophils stimulated with different chemoattractants.

The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis. Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet. The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface. The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth. In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant. The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant. This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632. The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth. Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors. The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants. The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8. The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.

ADP Ribose Transferases↗
PubMed1983

Radioimmunological measurements of total LH in sheep pituitary cells.

Procedures commonly used to extract LH from pituitary cells in order to measure total cell content were compared in four cell preparations. It was shown in 81 samples of cells suspended in 1 mM EDTA or 50 mM NaHCO3 that after freezing and thawing followed by any of a variety of treatments, there were no significant differences in the amounts of LH measured by RIA relative to the arbitrarily chosen reference treatment of vigorous pipetting. The additional treatments were multiple freezing and thawing, homogenisation, sonication, homogenisation in 25-100 mM Na2CO3 followed by rapid neutralisation, or none. The consistency of the results suggested that the same cellular pools of LH were being made accessible for measurement with all treatments. However, use of the more vigorous conditions of 1-2.5 M urea, 1% Triton X-100, or sonication on ice in 100 mM Na2CO3 decreased the amount of measurable hormone presumably due to its modification. In two cell preparations, homogenisation of cells in 100 mM Na2CO3 produced an additional 45% of measurable LH not accessible using other treatments nor from the source material in two other preparations.

Animals↗
PubMed1998

Sperm-oviduct epithelial cell monolayer co-culture: an in vitro model of sperm-female tract interactions in a marsupial, the tammar wallaby (Macropus eugenii).

Oviduct epithelial cell (OEC) monolayers were prepared from the isthmic and ampullary parts of the oviducts of FSH-primed tammar wallabies. Co-culture experiments found that 50-60% of wallaby spermatozoa attached immediately to OEC monolayers, tracheal cell monolayer controls, and the surface of culture dishes with and without Matrigel coating. Spermatozoa were considered to be attached if they remained on the culture surface after rapidly pipetting the co-culture medium five times. The percentages of attached and unattached spermatozoa were calculated from the number of spermatozoa recovered in the agitated supernatant. After 2 h co-culture the percentage of attached spermatozoa rose to 60-80%. After 6 h co-culture the number of spermatozoa attached to OEC monolayers derived from the oviductal isthmus remained high and only a small percentage were recovered in the agitated supernatant (unattached spermatozoa 3.85 +/- 0.76%, P = 0.67). However, after 6 h co-culture of spermatozoa with OEC monolayers derived from the ampulla and with the controls the percentage of attached spermatozoa declined significantly (unattached spermatozoa: ampullary monolayer 23.08 +/- 4.80%, P < 0.01; tracheal monolayer 23.23 +/- 5.18%, P < 0.01; Matrigel 27.23 +/- 7.76%, P < 0.01; plastic surface 28.19 +/- 5.30%, P < 0.01). After 6 h co-culture with ampullary and isthmic OEC monolayers, the percentage motility of both attached and unattached spermatozoa was maintained at 64.00 +/- 1.90% and 56.66 +/- 3.18% and 62.00 +/- 3.11% and 52.00 +/- 2.43%, respectively, and was then maintained at > or = 35% after 24 h incubation. In the controls, that is, tracheal monolayer and Matrigel, the motility of attached spermatozoa declined rapidly to 48.66 +/- 2.15% and 33.63 +/- 8.66%, respectively, at 6 h, and all spermatozoa were immotile after 24 h incubation. However, the motility of unattached spermatozoa in the controls (tracheal monolayer and Matrigel) was maintained at 57.33 +/- 3.00% and 34.54 +/- 9.27%, respectively, until 6 h and then declined rapidly, and all spermatozoa were immotile after 24 h incubation. Co-culture of wallaby spermatozoa with OEC monolayers also induced acrosomal modifications that were followed by acrosomal loss. At 6 h incubation 38.92 +/- 3.98% of spermatozoa on ampullary OEC monolayers and 36.50 +/- 3.81% spermatozoa on isthmic OEC monolayers had shed their acrosome. Acrosomal loss during co-culture with both isthmic and ampullary OEC monolayers was significantly (P < 0.01) greater than that observed on tracheal epithelial monolayer (24.42 +/- 1.90%, P < 0.01), Matrigel (20.70 +/- 2.71%, P < 0.01) and plastic (15.54 +/- 2.49%, P < 0.01). Co-culturing spermatozoa with OEC monolayers also induced a transformation from streamlined orientation of sperm head and tail to T-shaped (thumbtack) orientation in a small number (10-15%) of motile spermatozoa after 6 h incubation (data not shown). The significance of these results in relation to the role of the oviduct in sperm capacitation is discussed.

Acrosome Reaction↗
PubMed2005

The molecular diagnosis of porcine viral diseases: a review.

The worldwide occurrence and re-occurrence of transboundary diseases like foot-and-mouth disease or classical swine fever indicates that there is a high need for the development of powerful, robust and high-capacity new diagnostic methods, which are able to detect the causative agents before they could spread to large populations and cause tremendous losses. This article reports the experiences of a research group on the development of molecular methods for the improved diagnosis of a range of porcine viral diseases, including diseases on List A of the Office International des Epizooties (OIE). Nucleic acid hybridisation and various polymerase chain reaction (PCR) assays have been applied for routine diagnosis of a large range of viral diseases. During the last one-and-a-half decade more than 40 nested PCR assays have been developed to detect a variety of DNA and RNA viruses. False positive and negative results are avoided by the use of special tools, practices and internal controls of amplification (mimics). Recently, real-time PCR methods (TaqMan, molecular beacons, Primer-Probe Energy Transfer system) have been developed for the diagnosis of a wide range of diseases, such as foot-and-mouth disease, swine vesicular disease and vesicular stomatitis. Multiplex PCR packages have been developed for the simultaneous detection of eight important viruses of swine. By introducing nucleic acid extraction and pipetting robotics, together with the multi-channel real-time PCR machines, the diagnostic procedures have become rapid, robust and automated. In order to standardise the real-time PCR assays, the rules of OIE are considered. By following the five steps of OIE standardisation and validation, the new diagnostic procedures are nationally and internationally standardised and harmonised. The rapid, powerful and internationally standardised molecular diagnosis contributes to the reduction of losses caused by the transboundary viral diseases in swine populations.

Animals↗
PubMed1989

[Mycoplasma as a contaminant of cell cultures maintained in laboratories of private and official institutions].

Mycoplasma is one of the most serious contaminants of cell cultures. Its detection is very important in virology, as well as its eradication. The aim of this study was to verify the incidence of mycoplasma in cell lines maintained in seven laboratories of private, government and college institutions of the State of São Paulo, Brazil, for the purposes of research, production of reagents for diagnosis and production of biologicals for human and animal use. Of the 29 cell lines, eight were derived from human tissues and 21 from other animal species (dog, rabbit, mouse, hamster, monkey, pig, chicken and ox). Using the direct method with specific liquid and solid media for detection of mycoplasma, 48 out of the 106 cell samples tested were positive, corresponding to a contamination index of 45.28%. The incidence of contamination among the 35 cell samples of human origin was 51.43% (18 positive). Of the 71 samples originated from other species, 30 were positive (42.25%). The high incidence of contamination found calls for the adoption of measures for the prevention of this hazard: the elimination of mouth pipetting, the use of aseptic techniques and a rigid control of trypsin, serum and other components of cell culture media. The substitution of mycoplasma-free cultures for all contaminated ones and the performance of periodical tests for mycoplasma detection must also be carried out to prevent and avoid the dissemination of these organisms. Data obtained showed that contamination appeared in the 2nd (72.92%), in the 3rd (20.83%) and in the 4th passage (6.25%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Demineralization effect of EDTA, EGTA, CDTA and citric acid on root dentin: a comparative study.

The purpose of this study was to biochemically compare the decalcifying effects of 1% EDTA (pH 7.4), 1% EGTA (pH 7.4), 1% CDTA (pH 7.4), 1% citric acid solutions (pH 1.0 and 7.4) and saline solution (control) on root dentin. Forty-eight single-rooted teeth were used in this study. The canals were instrumented by the step-back technique and the roots were randomly divided into six equal experimental groups (n = 8) according to the irrigating agent tested. A total of 30 microL of each solution was pipetted into the root canal and allowed to set undisturbed for 5 minutes. After this time, 15 microL of the solutions were removed from each canal using a Hamilton syringe and placed in a container with 5 mL of deionised water. The microg/mL concentration of calcium ion (Ca2+) extracted from the root canal samples was determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES). Data were analysed by means of the Kruskal-Wallis and Mood's median tests. Citric acid solution at pH 1.0 removed more calcium than at pH 7.4 and than the other chelating solutions tested (p < 0.05). No differences were observed between EDTA and EGTA. Both EDTA and EGTA removed significantly more calcium than CDTA and citric acid at pH 7.4 (p < 0.05). There were no differences between citric acid at pH 7.4 and saline solution, which had the least efficacy for Ca2+ extraction (p > 0.05). These results indicate that citric acid at pH 1.0 is a good alternative as an irrigating solution to remove the smear layer and facilitate the biomechanical procedures.

Chelating Agents↗
PubMedDate not supplied

Surface plasmon resonance analysis to evaluate the importance of heparin sulfate groups' binding with human aFGF and bFGF.

Human acidic and basic fibroblast growth factors (aFGF and bFGF) are classic and well characterized members of the heparin-binding growth factor family. Heparin is generally thought to play an extremely important role in regulating aFGF and bFGF bioactivities through its strong binding with them. In order to unravel the mechanism of the interactions between heparin and FGFs, and evaluate the importance of heparin sulfate groups' binding with FGFs, surface plasmon resonance analyses were performed using IAsys Cuvettes System. Heparin and its regioselectively desulfated derivatives were immobilized on the cuvettes. aFGF and bFGF solutions with different concentrations were pipetted into the cuvettes and the progress of the interaction was monitored in real-time by Windows-based software, yielding kinetic and equilibrium constants for these interactions. In addition, in order to reduce the delicate difference among the cuvettes, inhibition analyses of mixture of FGFs and immobilized native heparin by modified heparins were also done. The data from these two methods were similar, indicating that all sulfate groups at 2-O, 6-O and N- in heparin were required for the binding to aFGF; and that their contribution to the binding was in the order 2-O, N- and 6-O-sulfate group. In contrast, definite contribution of the 6-O-sulfate group to the binding with bFGF was most apparent, while the other two sulfate groups appeared to be necessary in the order 2-O and N-sulfate group. These methods established here can be used for analysing the effect of sulfate groups in heparin on the binding with other human FGF members or other heparin-binding proteins.

Adsorption↗
PubMed1990

Inorganic mercury transport in the proximal tubule of the rabbit.

Inorganic mercury transport was studied in the S1, S2, and S3 segments of the isolated perfused proximal tubule of the rabbit. The concentration of mercury in the perfusate was 18.4 microM. At this concentration all three segments of the proximal tubule underwent degenerative changes that proceeded to cellular necrosis at the end of the tubule which was attached to the perfusion pipet. This pathological process progressed along the tubule for approximately 200 microns. The remainder of the tubule, to the collection pipette, remained intact and free of any pathological changes. In examining the transport of mercury under these condition, it was found that, on average, the S1, S2, and S3 segments all removed inorganic mercury from the luminal fluid at approximately 140 fmol min-1 mm-1. The transport of mercury, as measured by the appearance of 203Hg in the bathing solution, was 80% lower than the removal of 203Hg from the luminal fluid. The mercury appearing in the bath could be accounted for by passive leakage through the necrotic portion of the tubule in the S1 and S2 segments, but not in the S3 segment. Leakage could account for only 16.2% of the transepithelial movement of inorganic mercury in the S3 segment. Inorganic mercury taken up by the tubule (92%) was primarily associated with the structural proteins of the tubular epithelial cells, while very little (8%) was found in the tubular extract. The toxicity of inorganic mercury was determined by titration. Perfusion with 1 microM inorganic mercury produced necrosis. The pathological features appeared to be the same as those resulting with 18.5 microM inorganic mercury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Porous polysulfone coated with platelet-derived growth factor-BB stimulates proliferation of human periodontal ligament fibroblasts.

The purpose of this study was to investigate if the treatment of porous polysulfone (PPSF) with various concentrations of platelet-derived growth factor-BB (PDGF-BB) would stimulate the proliferation of the adherent human periodontal ligament fibroblasts (HPDLF) in culture. Sterilized PPSF cylinders were immersed in an Eagle's minimum essential medium supplemented with 0.5% fetal bovine serum and 1% penicillin/streptomycin containing either 0, 10, 20, or 50 ng/ml of PDGF-BB for 24 hours. After 24 hours, the PPSF cylinders were removed and allowed to dry then placed in culture plates for each time point. Pooled HPDLF (8 x 10(4)) and 3H-thymidine in medium were pipetted into each well to cover the treated and control PPSF cylinders and plates were then incubated. At 1, 4, and 10 days the PPSF cylinders were removed and macromolecular precipitation was performed. Incorporation of 3H-thymidine was measured and a 2-way ANOVA with repeated measures was performed. In addition, determination of binding and release was performed using I125-PDGF-BB treated PPSF at 0, 2, 12, and 24 hours, and at 4 and 10 days. Results showed that the effects on HPDLF were significant for dose (P = 0.0012; F = 5.74) and time (P = 0.0001; F = 40.83). At 4 days, the percent increases above the control for the doses 10, 20, and 50 ng/ml were 192%, 310%, and 162% respectively. In conclusion, our findings suggest that treating PPSF with PDGF-BB results in a significant increase in the proliferation of HPDLF cells adherent to PPSF.

Analysis of Variance↗
PubMed2002

The effects of varying degrees of allograft decalcification on cultured porcine osteoclast cells.

BACKGROUND: Demineralized freeze-dried bone allograft (DFDBA) is widely used in periodontal therapy as a scaffold for new bone formation in periodontal defects. It is demineralized, theoretically, to expose osteoinductive or osteoconductive bone matrix proteins that should facilitate osteogenesis. The degree of DFDBA demineralization varies between tissue banks and may affect clinical regeneration. A 2% residual calcium level in DFDBA has been shown to result in the highest alkaline phosphatase activity levels in cultured human periosteal cells and is optimally osteoinductive or osteoconductive for new bone formation. The purpose of this study was to evaluate the effect of 4 different residual calcium levels in commercially available DFDBA samples on porcine osteoclast activity as measured by resorption on calcium phosphate-coated disks. METHODS: Bone marrow was harvested from the femurs of 3-week-old farm pigs and cultured for 3 weeks. Hematopoietic stem cells were allowed to differentiate into mature active polykaryons displaying genuine osteoclast characteristics. The osteoclast cells displayed a dense actin band inside the margins of the cytoplasm under light microscopy. Culture media was decanted and collagenase added to free the attached cells. Equal cell samples were pipetted onto calcium phosphate-coated disks in 24-well plates. DFDBA samples with 1.44%, 2.41%, and 5.29% residual calcium; FDBA (30% residual calcium); and control cultures without allograft samples were prepared and all samples incubated for 1 week. Cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), Oregon Green 488-phalloidin, a stain for cytoskeletal proteins, and counterstained with propidium iodide. Specimens were examined by light and fluorescence microscopy using epi-illumination. Calcium phosphate disks were then rinsed in 5% sodium hypochlorite to remove adherent osteoclasts, and substrate surface changes were measured by white light interferometry and image analysis. RESULTS: A higher yield of TRAP-positive cells was produced without DFDBA; however, resorptive activity appears to be significantly increased in the presence of 2.41% residual calcium as compared to all other experimental groups (P<0.0065). CONCLUSION: In this in vitro model, porcine osteoclasts show significantly more resorptive activity as measured on calcium phosphate-coated disks in the presence of 2.41% residual calcium in DFDBA than in other DFDBA residual calcium levels.

Acid Phosphatase↗
PubMed2005

Surface modification of microchip input reservoirs for pressure-induced sample injection into microreactors.

Optimization of geometry and surface modification of microchip input reservoirs were performed to achieve uninterferenced pressure-induced sample injection of multiple samples into microreactors using a single syringe pump. Nine samples of 3.5 microL were pipetted onto input reservoirs and loading of PCR mixture into 260 nL microreactors was achieved followed by successful PCR amplification, confirming that no cross-contamination occurs during injection.

DNA↗
PubMed2003

Assessment of tear concentrations on therapeutic drug monitoring. III. Determination of theophylline in tears by gas chromatography/mass spectrometry with electron ionization mode.

A simple and sensitive method for the quantitation of theophylline (TP) in tears and plasma developed using gas chromatography/electron-impact ionization/mass spectrometry. Tears were collected by non-invasive Schirmer method. Plasma was pipetted on a Schirmer tear test strip (cutting to 5 mm x 5 mm). Then, TP was converted directly into its pentafluorobenzoyl amide derivative without the need to perform any extraction from the biological fluid absorbed on Schiemer test paper and was quantified by gas chromatography-selected ion recordings with electron ionization mode. The concentrations in tears [C]t correlated very well with those of the free form in the plasma [Cf]p and those of the total form in the plasma [Cb+f]p. The ratios between TP concentrations in tears and plasma (free and total form) were as follows: [C]t/[Cb+f]p=0.53+/-0.20; [C]t/[Cf]p=1.21+/-0.19; [Cf]p/[Cb+f]p=0.44+/-0.14. The ratios of [C]t/[Cb+f]p, [C]t/[Cf]p and [Cf]p/[Cb+f]p were in good agreement with the previously published data.

Journal Article↗
PubMed2000

Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis.

In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.

Automation↗
PubMed2000

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA.

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.

DNA↗
PubMed2001

High-resolution semi-quantitative real-time PCR without the use of a standard curve.

The repeatability and sensitivity of a simple, adaptable, semi-quantitative, real-time RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the LightCycler real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.

Animals↗