Eimeria tenella (Eucoccidiorida): a quantitative assay for sporozoite infectivity in vivo

A quantitative technique for the assessment of sporozoite infectivity in vivo, using intra-cecal inoculation of Eimeria tenella sporozoites, has been developed. Evaluation of the infection using cecal lesion scores and oocyst counts showed that this technique should be useful for the quantitation of sporozoite viability and thus for the anti-sporozoite activity of different treatments prior to inoculation. Pre-treatment of sporozoites with heat-inactivated hyperimmune antisera neutralized sporozoite infectivity in vivo and indicated that antibodies in the absence of complement inhibited sporozoite infectivity in vivo.     

A prestaining method for the quantitative assay of proteins on polyacrylamide gels

Dyes have been synthesised¹, which make it possible to prestain proteins prior to electrophoresis on polyacrylamide gels. After discussing the criteria which have to be fulfilled by the dyes, their method of application is described. The method has been tested on a number of selected acidic and basic proteins and also on peptide obtained by the digestion of bovine serum albumin with cyanogen bromide. Excellent reproducibility, stoichiometry and a sensitivity of 0.2 μg with some proteins has been obtained.     

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.     

Anisakis pegreffii: a quantitative fluorescence PCR assay for detection in situ

To facilitate improved diagnosis and detection of the third stage larva (L3) of Anisakis pegreffii from the Minnan-Taiwan bank fishing ground in Taiwan Strait, a real-time PCR method for the detection in situ and differentiation was developed to amplify a region of the second internal transcribed spacer (ITS-2) of this parasite. The real-time PCR assay was capable of detecting 1/3 of a single L3 in 30 mg of marine fish tissue, and also exhibited a high level of specificity for A. pegreffii, no fluorescence signals were observed in other five major larval anisakid species found in commercial marine fishes caught in this fishing ground.     

A rapid and reproducible assay for quantitative estimation of proteins using bromophenol blue

A method for the quantitation of proteins in solution which involves the binding of bromophenol blue to proteins under acidic conditions has been developed. The binding of the dye to proteins is accompanied by the appearance of a strong absorbance at 610 nm, which is almost linear over the range of 10 to 80 μg for the seven proteins studied. The absorbance at 610 nm can be measured immediately after the mixing of the protein and dye solutions and is stable over a period of 8 hr. The method has very few interferences, most of which can be corrected for by the use of proper controls. Phenol, sodium dodecyl sulfate, and Triton X-100 (the last with some error) may be used with this assay at concentrations that produce strong interferences with similar methods using Coomassie brilliant blue G-250.     

A facile,sensitive and quantitative membrane‐binding assay for proteins

Soluble proteins that bind membranes function in numerous cellular pathways yet facile, sensitive and quantitative methods that complement and improve sensitivity of widely used liposomes‐based assays remain unavailable. Here, we describe the utility of a photoactivable fluorescent lipid as a generic reporter of protein‐membrane interactions. When incorporated into liposomes and exposed to ultraviolet (UV), proteins bound to liposomes become crosslinked with the fluorescent lipid and can be readily detected and quantitated by in‐gel fluorescence analysis. This modification obviates the requirement for high‐speed centrifugation spins common to most liposome‐binding assays. We refer to this assay as Proximity‐based Labeling of Membrane‐Associated Proteins (PLiMAP).     

A quantitative assay for poly(ethylene glycol) without interference by proteins

A method is described for the quantitative determination of poly(ethylene glycol) having an average molecular weight of ≥400. The method is based on a measurement of the intensity of light which is seattered by the turbid suspension produced by addition of the polymer to Nessler's solution. Small amounts of plasma proteins (0.001 mg/ml) drastically diminished the turbidity. This interference can be eliminated by prior adsorption of the protein on Al(OH)₃ gel.     

An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy

We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported.     

Electrochemistry of neuropeptides: a possible method for assay and in vivo detection

The electrochemical activity of catechol and indoleamines has been utilized to develop sensitive assays for amine neuro-transmitters and metabolites. We present evidence that several neuropeptides are electrochemically active and that this property may be used to develop peptide assays similar in sensitivity to those available for the amines. The electrochemical activity of twenty synthetic neuropeptides and their constituent amino acids were tested in buffered solutions using differential pulse voltammetry (DPV) and micro-carbon paste working electrodes. The studies show that peptides which are electroactive include Met-and Leu-enkephalin, the cholecystokinins (CCK-8, CCK-4), caerulein, neurotensin, gonadotrophin releasing hormone (LH-RH), α-melanocyte stimulating hormone (αMSH), somatostatin and vasopressin. The electrochemical oxidation potentials of these peptides are distinct and separable from those of the amines and are apparently associated with the presence and combination of specific amino acids (tyrosine, tryptophan and cysteine) in the peptide sequence. DPV $\text{in vitro}$ was used to demonstrate the presence of 5-hydroxytryptamine and caerulein in extracts of amphibian skin. With electrodes implanted in the rat striatum DPV $\text{in vivo}$ exhibited oxidation potentials which may relate to neuropeptide oxidation.     

Fluorescent proteins as a toolkit for in vivo imaging

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.     

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.