A widely applicable analytical system for biological stains: reverse-phase thin layer chromatography

The suitability of reverse-phase thin layer chromatography using a commercial adsorbant and aqueous methanol as an analytical tool for biological stains was investigated. The wide range of applicability of this technique is shown by the fact that of 120 dyes used as biological stains, 84 of diverse chemical character were successfully chromatographed by varying only the water content of the eluent. Unsuccessful chromatography was due either to immobility or streaking. Dyes exhibiting this behavior can be identified prior to chromatography by inspection of their structural formulae. Rf values were found to be significantly correlated with the calculated partition coefficients. This relationship provides information for the identification of dye components revealed by chromatography and a discussion of its use in the chemical characterization of various dyestuffs is presented.     

High-throughput screens for fluorescent dye discovery

A recent screen of a combinatorial library of fluorescent compounds discovered fluorescent dyes that were able to distinguish myoblasts from differentiated myotubes. New fluorescent dyes that respond to biologically relevant changes in cell state or type are useful as stains in a wide variety of biological experiments, including high-throughput screens for chemical and genetic regulators. Combining this approach with microscopy imaging is likely to be even more powerful and might lead to the discovery of new dyes with interesting and useful properties.     

Analysis and Purification of Biological Stains by Gel Filtration

Some sixty biological stains of widely varying type have been subjected to gel filtration chromatography in columns of Sephadex LH-20 (Pharmacia, Uppsala) resin swollen by dimethylformamide saturated with NaCl. Most dyes contained more than one coloured constituent. Measures of their molecular weights were obtained. The use of the method for analysis was as effective as other common chromatographic or electrophoretic procedures but was technically simpler. Preparative use of the method merely involved using a larger column of gel, though occasionally use of ethanol as solvent gave better resolutions and did provide easier recovery of separated dyes.     

Comparative Absorption Readings Obtained with Spectrophotometers of Various Types

One of the most important means of identifying a dye is by spectrophotometry. This method has been used as a criterion for judging the samples of stains submitted by manufacturers ever since the certification plan for biological stains was adopted. All specifications that have been drawn up for biological stains, such as those given in the 7th edition of the National Formulary, have included spectrophotometry requirements.     

An Assessment of Routine Chromosomal Staining Procedures in Radioautography

Unlabeled human chromosome preparations were treated with commonly employed chromosome stains as follows: (I) they were stained, destained, coated with liquid emulsion, developed, fixed, and restained; (II) stained and coated directly; or (III) coated and then stained. Of the stains tested, the methylene blue-eosin type (Giemsa, MacNeal's, Wright's) was useful for application after coating, although a similar stain (eosin-Stevenel's blue) caused formation of a heavy precipitate in the emulsion when so used. None of these stains could be employed before coating, however, even though they were removed with acid alcohol prior to dipping, because they caused chemographic grain formation in the emulsion. Aceto-orcein and Feulgen could not be employed after coating because the procedures removed the emulsion from the slides. Safranin was also found to be ineffective for staining coated preparations due to chemical changes caused by the photographic processing. The only stain which did not cause chemography, and hence can be used before coating slides, is aceto-orcein. Since this stain fades during radioautographic processing and cannot be employed after coating, we recommend secondary use of one of the methylene blue-eosin type stains for revisualization of the chromosome spread.     

Absorption of protamine-insulin in diabetic patients. I. Preparation and characterization of protamine-125I-insulin.

Protamine-125I-insulin with low specific radioactivity was prepared using 125, 127I-insulin, 0.2 I/mole. The preparations were characterized by disc electrophoresis, isoelectric focusing and analytical gel chromatography in order to evaluate the suitability of 125I-insulin as marker for insulin in protamine-insulin. The stability of the preparations was followed up to 90 days at 4 degrees C. The biological and immunological activity was determined in mice, isolated rat fat cells and by radioimmunoassay. It was concluded that both from a chemical and a biological point of view, the protamine-125I-insulin is a satisfactory preparation that might be used in absorption studies.     

The value of simple lipid stains for typing skeletal muscle fibres

Summary Skeletal muscle fibre types can be distinguished rapidly with simple lipid stains. Comparative studies showed that Sudan Black B is superior to Oil Red O for this purpose and that optimum staining is obtained using unfixed sections or sections fixed in calciumglutaraldehyde. Factors that possibly influence the staining reaction, such as freeze-thawing, are considered. The stained lipids were identified by thin layer chromatography.     

Improving Biological Dyes and Stains: Quality Testing Versus Standardization

This paper discusses the impact of both standardization and quality testing of dyes and stains in biology and medicine. After a brief review of why standardized dyes and stains are not presently available commercially, two types of testing and ways of improving dye quality are described. National or international organizations could be established to define standardization of dyes and stains. Standardization would be specifically defined as a list of physico-chemical parameters such as elaborated in this paper. Commercial batches of comparable quality may be labeled by the supplier as “standard dye.” a procedure currently performed by the European Council for Clinical and Laboratory Standardization (ECCLS). Also recommended to improve dye quality is commercial dye testing by independent laboratories with subsequent certification for use. This sort of quality control is currently carried out in the United States by the Biological Stain Commission (BSC). The advantages and disadvantages of both techniques and the use of image analysis for the definition of standards are discussed. A combination of both the BSC testing protocols and the ECCLS standards should be established for extended quality control of biological dyes and stains.     

High-resolution melt analysis of DNA methylation to discriminate semen in biological stains

The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.     

Chemical synthesis of La1 isolated from the venom of the scorpion Liocheles australasiae and determination of its disulfide bonding pattern

La1 is a 73‐residue cysteine‐rich peptide isolated from the scorpion Liocheles australasiae venom. Although La1 is the most abundant peptide in the venom, its biological function remains unknown. Here, we describe a method for efficient chemical synthesis of La1 using the native chemical ligation (NCL) strategy, in which three peptide components of less than 40 residues were sequentially ligated. The peptide thioester necessary for NCL was synthesized using an aromatic N‐acylurea approach with Fmoc‐SPPS. After completion of sequential NCL, disulfide bond formation was carried out using a dialysis method, in which the linear peptide dissolved in an acidic solution was dialyzed against a slightly alkaline buffer to obtain correctly folded La1. Next, we determined the disulfide bonding pattern of La1. Enzymatic and chemical digests of La1 without reduction of disulfide bonds were analyzed by liquid chromatography/mass spectrometry (LC/MS), which revealed two of four disulfide bond linkages. The remaining two linkages were assigned based on MS/MS analysis of a peptide fragment containing two disulfide bonds. Consequently, the disulfide bonding pattern of La1 was found to be similar to that of a von Willebrand factor type C (VWC) domain. To our knowledge, this is the first report of the experimental determination of the disulfide bonding pattern of peptides having a single VWC domain as well as their chemical synthesis. La1 synthesized in this study will be useful for investigation of its biological role in the venom. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Chromatograms of Biological Stains on Acid and Basic Adsorbents

A simple method of preparing chromatograms of mixtures of various biological stains on common acid and basic adsorbents is described. From the study of such chromatograms, three kinds of preferential adsorption can be recognized. (1) Due to the preferential adsorption of acid stains on basic adsorbent, a mixture of stains will separate itself into successive color bands on the chromatogram according to their individual acidity or basicity. The chromatogram, therefore, of a mixture of three stains—acid, neutral and basic—will appear on basic adsorbent according to the above order. The reverse order results if the acid adsorbent is used. The stains can be taken off from the adsorbents by shaking with proper solvents. (2) The components of some neutral stains or a mixture of some similarly charged stains can also be separated in a chromatogram. The order of appearance seems to be independent of the acid or basic nature of the adsorbents. This is similar to the chromatogram of leaf extract of plant pigments. (3) Finally, some mixtures of acid and basic stains do not follow their regular sequence of appearance on the chromatogram when adsorbed on magnesia. The reason for this anomaly is not clear, probably due to formation of adsorption complex.

This technic can be used to detect and separate mixtures of stains and to demonstrate the nature of adsorption and theory of staining. It can be used also as a preliminary test for the choice of solvent and adsorbent for chromatographic analysis. For the purpose of demonstration, an artificial cell can easily be made by impregnating talc (acid) and magnesia (basic) in collodion, in the form of nucleus, cytoplasm, etc., which is stained by following the general histological technic after the collodion is dried.     

Mycological and FTIR analysis of biotic foxing on paper substrates

The small rusty stains (foxing) frequently found on historic paper documents, books, and prints have generally been analysed in the past by optical microscope through their morphochromatic appearance under visible light and UV radiation. Despite increased research efforts with more sophisticated techniques (mainly SEM and XRF), the biotic or even chemical origin of these stains remains unclear. The purpose of this paper is to verify to what extent a simple technique such as FTIR-ATR spectroscopy can be utilised for a clearer understanding of the controversial nature of foxing. Since this technique is sensitive to several organic chemical groups that are in common with both fungi and gelatine-sized ancient paper, some modern cardboards stained by biotic foxing have been selected for the analyses. The results clearly show the importance of FTIR and mycological analyses for the identification of residual microfungal agents, together with the by-products of their activity on paper substrates.     

alpha-Zearalenol, a major hepatic metabolite in rats of zearalenone, an estrogenic mycotoxin of Fusarium species

The chemical and biological properties of the hepatic metabolite of zearalenone, an estrogenic and non-steroidal fungal toxin produced by Fusarium species, were investigated by employing TLC, GC/MS, high pressure liquid chromatography and fluorospectral analyses, as well as uterine weight bioassay in immature mice. All the chemical and physical data supported the view that the major metabolite, obtained by incubating zearalenone with S-9 and microsomes of rat liver in the presence of NADPH, is C-6'-alpha-hydroxylated zearalenone (alpha-zearalenol). The estrogenic activity of this metabolite was several times higher than that of the parent zearalenone, and the results of biological and toxicological evaluations of alpha-zearalenol are discussed.     

A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

A Brief History of the Biological Stain Commission: its Founders,its Mission and the First 75 Years

The need for batch-to-batch consistency in available dyes and stains used for biological purposes posed a considerable problem for United States scientists following World War I. Prior to that time, most of the acceptable stains in this country were of German origin. In an attempt to standardize the performance of biological stains and dyes, the Society of American Bacteriologists in 1922 appointed Dr. Harold Conn to form the Committee on the Standardization of Biological Stains. To assist him, Dr. Conn recruited scientists from several major professional scientific societies. Mr. Holland Will, a Rochester, NY, vendor of stains, was also instrumental in the Committee's success. This article traces the origin, mission and accomplishments of the product of that Committee, the Biological Stain Commission, through the past 75 years, and focuses on some of the major events that influenced and shaped its development.     

A Brief History of the Biological Stain Commission: its Founders, its Mission and the First 75 Years

The need for batch-to-batch consistency in available dyes and stains used for biological purposes posed a considerable problem for United States scientists following World War I. Prior to that time, most of the acceptable stains in this country were of German origin. In an attempt to standardize the performance of biological stains and dyes, the Society of American Bacteriologists in 1922 appointed Dr. Harold Conn to form the Committee on the Standardization of Biological Stains. To assist him, Dr. Conn recruited scientists from several major professional scientific societies. Mr. Holland Will, a Rochester, NY, vendor of stains, was also instrumental in the Committee's success. This article traces the origin, mission and accomplishments of the product of that Committee, the Biological Stain Commission, through the past 75 years, and focuses on some of the major events that influenced and shaped its development.     

Phosphoproteomics strategies for the functional analysis of signal transduction

Protein phosphorylation is a key regulatory mechanism of cellular signalling processes. The analysis of phosphorylated proteins and the characterisation of phosphorylation sites under different biological conditions are some of the most challenging tasks in current proteomics research. Reduction of the sample complexity is one major step for the analysis of low-abundance kinase substrates, which can be achieved by various subcellular fractionation techniques. One strategy is the enrichment of phosphorylated proteins or peptides by immunoprecipitation or chromatography, e.g. immobilised metal affinity chromatography, prior to analysis. 2-DE gels are powerful tools for the analysis of phosphoproteins when combined with new multiplexing techniques like DIGE, phosphospecific stains, autoradiography or immunoblotting. In addition, several gel-free methods combining chromatography with highly sensitive MS have been successfully applied for the analysis of complex phosphoproteomes. Recently developed approaches like KESTREL or 'chemical genetics' and also protein microarrays offer new possibilities for the identification of specific kinase targets. This review summarises various strategies for the analyses of phosphoproteins with a special focus on the identification of novel kinase substrates.     

Enzymatic production of epigallocatechin by using an epigallocatechin gallate hydrolase induced from Aspergillus oryzae

Catechins are a group of polyphenolic compounds that are antioxidants having beneficial biological activities. There are four main catechins in green tea, and each has its own biological features. In order to fully exploit prominent biological activities of specific catechins and to develop new medicine from catechins, it is necessary to obtain pure catechin preparations by isolation from natural sources, by chemical synthesis, or by biotransformation reactions with high yield and specificity. In this study epigallocatechin gallate (EGCG) can be hydrolyzed to epigallocatechin (EGC) by a hydrolase from Aspergillus oryzae after induction by addition of EGCG to the cultures. However, cultures without EGCG induction did not show any EGCG hydrolysis activity. The yield of EGC could reach at least 70%. Thin layer chromatography and high performance liquid chromatography were applied to separate and quantify EGCG and EGC.     

Screening for N-glycosylated proteins by liquid chromatography mass spectrometry

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.     

Studies on Textile Dyes for Biological Staining: I. Pontacyl Blue Black SX, Pontacyl Violet 6R and Luxol Fast Yellow TN

After a series of commercial grade textile dyes were screened, three -were found to be excellent biological stains. Two of these, pontacyl blue black SX and pontacyl violet 6R, were nuclear stains, the best results being obtained after mordanting with chrome salts. The third dye, luxol fast yellow TN, imparted a brilliant yellow stain to the cytoplasm and collagen fibers when used in alcoholic solution. A technical procedure for the use of either of the nuclear stains combined with the yellow cytoplasmic stain is given.