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PubMed1985

Why does Coomassie Brilliant Blue R interact differently with different proteins? A partial answer.

Dimethyl sulfoxide was found to be effective for extraction of Coomassie Brilliant Blue R-250 (Coomassie R) from stained proteins on polyacrylamide gel slices. A good correlation was found between the ability of different proteins to bind Coomassie R and their capacity for interaction with Coomassie Brilliant Blue G-250 (Coomassie G) in solution. Scatchard analysis showed that the number of Coomassie R ligands bound to each protein molecule is approximately proportional to the number of positive charges on the protein, about 1.5-3 dye molecules/charge.

Electrophoresis, Polyacrylamide Gel↗
PubMed2003

Photocatalytic degradation of two selected textile dye derivatives, eosine yellowish and p-rosaniline, in aqueous suspensions of titanium dioxide.

Thephotocatalytic degradation of two selected textile dye derivatives, eosine yellowish (1) and p-rosaniline (2) has been investigated in aqueous suspensions of titanium dioxide under a variety of conditions. The degradation was studied by monitoring the change in substrate concentration employing UV spectroscopic technique and decrease in Total Organic Carbon (TOC) content as a function of irradiation time under a variety of conditions. The degradation of the dye was studied under different conditions such as pH, catalyst concentration, substrate concentration, different types of TiO2 and in the presence of electron acceptor such as hydrogen peroxide (H2O2), potassium bromate (KBrO3), and ammonium persulphate (NH4)2S2O8) besides molecular oxygen. The degradation rates were found to be strongly influenced by all the above parameters. The photocatalyst Degussa P25 was found to be more efficient as compared with other photocatalyst in the case of p-rosaniline, whereas UV100 was found to be better for the degradation of eosine yellowish. The xanthene dye (1) was found to degrade faster as compared to the triphenylmethane dye (2). The degradation products were analyzed by GC/MS technique and plausible mechanism for the formation of products have been proposed.

Catalysis↗
PubMed2005

Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green.

Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.

Acrylic Resins↗
PubMed1996

Microfluorometric study of nuclear DNA in normal human epidermis.

Quantitative and qualitative changes of nuclear DNA were analyzed by microfluorometry in normal human epidermis during terminal differentiation. Normal human epidermis from five healthy volunteers was separated by an EDTA-trypsin method and fractionated by Percoll density gradient. Staining was carried out with either Feulgen reaction, Acridine Orange or 4'-6-diamidino-2-phenylindole (DAPI). When the cells with lower density (granular layer cells) were stained with Acridine Orange or DAPI, the greatest population was found at lower value of DNA than that in the cells stained with Feulgen reaction. After thermal denaturation procedure, a ratio of double-stranded DNA to single-stranded DNA was highest in the cells with lower density. These results seem to suggest the qualitative and quantitative changes of nuclear DNA during terminal differentiation of human epidermal keratinocytes.

Acridine Orange↗
PubMed2003

Fluorescent peptide probes for high-throughput measurement of protein phosphatases.

A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide was found to be sensitive to the phosphorylation status of an adjacent threonine residue. Upon complete dephosphorylation of the dye-labeled phosphopeptide, a 56% decrease in fluorescence intensity was observed. The change in fluorescence was correlated with the release of inorganic phosphate from the phosphopeptide as measured using the malachite green assay. Conjugation of the fluorophore to the phosphopeptide was found to have no adverse effect on catalysis. A series of four phosphopeptide substrates were developed and characterized to probe PP1 and PP2A activity. The optimum phosphopeptides were then used to determine inhibition parameters for three natural protein phosphatase inhibitors. The use of a peptide-based approach has introduced a degree of specificity not observed with many conventional phosphatase substrates, while retaining the advantages of a real-time homogeneous fluorescence-based format, making the assay ideal for high-density screening.

Enzyme Inhibitors↗
PubMed2001

Lessons from a proficiency testing event for acid-fast microscopy.

OBJECTIVES: To evaluate the routine performance and the technical parameters of different acid-fast staining methods: Kinyoun, Ziehl-Neelsen (ZN), auramine, and auramine-rhodamine. DESIGN AND PARTICIPANTS: The performance of 167 laboratories was analyzed using prestained and unstained slides. SETTING: Laboratories holding New York State permits. RESULTS: The results revealed that Kinyoun's cold carbol fuchsin method is inferior to both the ZN and fluorochrome (auramine and/or auramine-rhodamine) methods. Even though 91% of the participants used commercial staining kits, the study identified unexpected errors concerning the concentration of carbol fuchsin, time for staining and counterstaining, and the concentration of acid alcohol for decolorization, which may significantly influence the sensitivity. Besides these findings, the present study showed that the examination of < 300 view fields may also decrease the sensitivity of acid-fast microscopy. In addition, we found that the sensitivity and specificity of the ZN and fluorochrome methods are comparable if the procedural standards are followed. CONCLUSIONS: The strict and ongoing quality control of the "simple to perform" acid-fast microscopy and the immediate review of commercially available staining kits are necessary. Because of the rapidity of the fluorochrome method, laboratories with large specimen numbers should use this technique. In all other cases, the ZN method should be used. Moreover, all clinicians should be aware of the method of acid-fast microscopy used and the proficiency of the laboratory in performing the assay.

Benzophenoneidum↗
PubMed2000

Detection of DNA in ancient bones using histochemical methods.

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

Animals↗
PubMed2000

DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists.

Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp. and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex. This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body. Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g. eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures. These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts. These features are characteristic of their protist class (Parabasalia). The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all. The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI. Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence. I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes. The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.

Acridine Orange↗
PubMed1993

Bradford protein assay and the transition from an insoluble to a soluble dye complex: effects of sodium dodecyl sulphate and other additives.

The addition of sodium dodecyl sulphate (SDS) (0.0015-0.006%), phenol (0.25-0.5%) or sodium hydroxide (0.025-0.1 M) to the Bradford dye reagent does not improve the solubility of the Coomassie blue-protein dye complex. Centrifugation of the assay tubes, 10 min after the addition of reagent, results in complete loss of colour yield as indicated by the absorbance (A595) of the recovered supernates. At protein-concentrations above the working range of the assay, centrifugation indicates a transition from an insoluble to a soluble protein-dye complex. This transition is characteristic of an individual protein and is influenced by assay modification. Low protein concentrations appear to provide nucleation sites for precipitation of Coomassie blue whilst higher protein concentrations increase its solvation. A soluble dye chromophore is only formed above the working range of the assay indicating that precipitation of the dye by protein contributes to the assay mechanism.

Centrifugation↗
PubMed1987

Effect of alternating current exposure on the resistivity of resting Escherichia coli B cells to crystal violet and other basic dyes.

Phosphate buffer suspensions of resting Escherichia coli B cells at pH 7.0 were anaerobically exposed to alternating current (a.c.) of 50 Hz at a current density of 600 +/- 60 mA/cm2 and 34 degrees +/- 3 degrees C. The minimum inhibitory concentrations of eight basic dyes: crystal violet, malachite green, brilliant green, fuchsin, methylene blue, toluidine blue, safranin and acriflavine for exposed cells were decreased to about the half values of those for unexposed ones when both cells were grown in the minimal medium including one of the dyes. The integrated viabilities of exposed cells tended to decline with increasing concentration of the dyes markedly more than those of unexposed ones, whereas the exposed cells took up the dyes less readily than the unexposed cells. These results suggested that a.c. exposure may serve as an agent which renders E. coli cells susceptible to the basic dyes.

Coloring Agents↗