Dye Staining Method Research Articles

Evans Blue Staining Method for Detection of Proteins on Polyacrylamide Gels with Rhodamine B

Abstract A new staining method employing a mixed dye technique is simple, rapid, and sensitive. Solutions of Evans blue (EB, 0.01 %) and rhodamine B (RB, 0.5 %) were mixed up with 1:1 volume ratio. This mixed dye staining method detects as little as 10 ng of bovine serum albumine (BSA). EB is used for protein staining based on its structural similarity to Coomassie brilliant blue R–250 (CB). RB acts as a supplementary agent which inhibits the binding of EB to gel matrix, and so increases the staining effect of EB and enables to achieve cutting down staining/destaining times.

A stereophotogrammetric method for determining in situ contact areas in diarthrodial joints, and a comparison with other methods

Determination of contact areas in diarthrodial joints is necessary for understanding the state of stress within the articular cartilage layers and the supporting bony structures. The present study describes the use of a stereophotogrammetry (SPG) system [Huiskes et al., J. Biomechanics , 18, 559–570 (1985) and Ateshian et al., J. Biomechanics, 24, 761–776 (1991)] for determining contact areas in diarthrodial joints, using a surface proximity concept similar to the one used by Scherrer et al. [ ASME J. biomech. Engng, 101, 271–278 (1979)]. This method consists of evaluating the proximity of the articular surfaces to determine joint contact areas using precise geometric models of the joint surfaces obtained from the SPG system, and precise kinematic data, also obtained from SPG. In this study, the SPG method for determining contact areas is compared to other commonly used methods such as dye staining, silicone rubber casting and Fuji film contact measurement techniques which have been often used and reported by other investigators. The bovine glenohumeral joint and the bovine lateral tibiofemoral articulation (without the meniscus) were used to represent congruent and incongruent joints, respectively. While all the methods yielded consistent contact patterns for the incongruent tibiofemoral articulations, the results for the congruent bovine glenohumeral joints showed that the SPG and Fuji film methods were in better agreement than those obtained from the dye staining and silicone rubber casting methods. The advantages of the new SPG method are that it can be used for intact joints, and used repeatedly and quickly thus making contact-area movement analyses possible [Soslowsky et al., J. orthop. Res., 10, 524–534 (1992)]. The results of this comparison study show that the SPG technique is a reliable and versatile method for determining contact areas in diarthrodial joints.

Native and degraded forms of sweet potato starch phosphorylase.

By immunoblotting using monoclonal and polyclonal mouse antibodies against sweet potato (Ipomoea batatas (L.) Lam.) starch phosphorylase (SP), and by activity and dye staining methods, fresh extracts obtained from sweet potatoes immediately after harvest or from those being stored under various conditions were analyzed for SP by PAGE. Freshly prepared pure or crude SP, or those stored under various conditions were also analyzed. We found that the native enzyme from either of the cultivars Tainong 57 or 65 was 220-kDa in size and constituted of two 112-kDa subunits. Either in vivo or in vitro, storage caused degration of the enzyme to two groups of small subunits of about 50-kDa each. On prolonged cold storage of purified enzyme in 50% glycerol, the formation of a small amount of a 250-kDa protein was detected. It had a 150-kDa subunit that was formed presumably by transpeptidation catalyzed by a contaminating protease. This 250-kDa form had the primed SP activity only. The SP from the two kinds of cultiva...

A Nuclear Marker for Mammalian Cells and Its Use with Intracerebral Transplants

The Hoechst dye staining method has been successfully applied to the central nervous system in mammals and its use has been demonstrated in intracerebral transplantation. The technique is rapid, simple and based on intrinsic nuclear properties. It was found to be permanent and valid whatever the animal strains or ages, allowing the distinction of rat cells from those of mouse, studied either separately or in a cross-transplantation model. It permitted the detection of grafted cells in the area of transplantation and the observation of early dispersion around the implantation site. Moreover, it can be combined with immunohistochemistry as demonstrated by a myelin marker in a relevant model. Immunodetection can thus help to directly observe grafted cells, at distance from the locus of transplantation, confirming their presence in the graft-type myelin patches. Because of its rapid performance, this technique can be used systematically after transplantation to check for the presence of grafted cells in the host.

Enzymoblotting: Visualization of electrophoretically separated enzymes on nitrocellulose membranes using specific substrates

AbstractEnzymoblotting is a technique for the detection of electrophoretically separated enzymes after transfer to nitrocellulose (NC) membranes and subsequent incubation with suitable substrates. After agarose gel electrophoresis or isoelectric focusing in ultrathin polyacrylamide gels, blotting of the separated proteins/enzymes to NC membranes was performed by capillary forces for 30 min. Hydrolases, e. g. pancreatic proteinases, were visualized using amino acid/peptide substrates based either on the β‐naphthol or para‐nitroanilide (pNA) group. Both types of substrates were used with good results, but the pNA substrates provide greater specificity and sensitivity. An oxidoreductase, lactate dehydrogenase, was visualized using an electron transfer dye staining method. Enzymoblotting also allows detection of inactive proenzymes since conversion to active enzymes in situ on the NC membrane may be performed by pre‐incubating the NC membranes with activation enzymes. The enzymoblotting technique offers several advantages over traditional gel staining methods. The enzymes are immobilized on the NC membrane, preventing diffusion during substrate incubation, and as a result, high resolution is retained even after long incubation times; the product obtained as a result of the enzyme activity can be chemically derivatized and bound to the membrane, thus widening the categories of possible substrates; subsequent incubation of the same NC membrane in various substrate solutions can be performed; enzymoblotting can be used in combination with other techniques, e. g., immunoblotting for parallel structural and functional studies.

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

A quantitative determination of the relative amount of histones in polyacrylamide gel by silver stain

On the basis of scanning densitometry of the stained gel, the conditions for the quantitative determination of individual histones by silver was examined and compared with the dye-staining method, in terms of higher sensitivity and faithful quantitation. Fixation with formaldehyde, coupled with simultaneous prestaining with Coomassie brilliant blue (CBB), was found to be most suitable. Prior fixation in acidic alcohol alone failed to stain the histones accurately, but this failure could be partly alleviated by prestaining with CBB. Although the sensitivity for detecting histones by silver staining is lower than that for neutral proteins by about 10-fold, it is at least 10-fold higher than the CBB stain.

Molecular cloning and expression of a Bacillus subtilis β-glucanase gene in Escherichia coli

A Bacillus subtilis gene coding for an endo-β-1,3-1,4-glucanase has been transferred to Escherichia coli by molecular cloning using bacteriophage λ and plasmid vectors. The gene is contained within a 1.6-kb EcoRI- PvuI DNA fragment and directs the synthesis in E. coli of a β-glucanase which specifically degrades barley glucan and lichenan. A novel dye-staining method has been developed to detect β-glucanase activity in colonies on agar plates.

Identification of paraneurons by labelling with quinacrine (Atebrin).

Quinacrine (Atebrin), an antimalarial drug that binds to adenine nucleotides, was used to label paraneurons. The drug was dissolved in physiological saline and intraperitoneally injected (rats, mice, guinea pigs) in concentrations of 120 or 200 mg/kg body weight. The animals were killed three days later and paraneurons were studied in the fluorescence microscope aftre freeze drying. Bright fluorescence is observed in adrenomedullary cells, SIF cells, carotid body chief cells, pancreatic islet cells, anterior pituitary cells, pinealocytes, and mast cells. Weak fluorescence occurs in thyroid parafollicular Merkel cells, and a few entero-endocrine cells. It is suggested that quinacrine binds to ATP or related adenine nucleotides which are stored in secretory granules of paraneurons. Varying fluorescence intensity seems to depend on different concentrations of adenine nucleotides within the storage granules, as well as on the different size and number of these granules in various paraneurons. Besides formaldehyde-induced fluorescence of biogenic amines, HCl-basic dye staining methods and electron microscopy, the method is useful to identify and define paraneurons.

An Arterial Injection Method through Inferior Thyroid Artery for the Treatment of Laryngeal Cancer

We have tried to apply anti-cancer drugs by the method of arterial injection to inferior thyroid artery for the treatment of the laryngeal cancer. The patient was a 66-year-old Japanese male with the cancer mainly located in the posterior commissure of the larynx. After truncus thyreocervicalis and its branches were exposed, the Hakko's teflon tube with the outside diameter 1mm was retro-catheterized through transversa colli artery. We are always mindful of that an anatomical variety exists on truncus thyreocervicalis and inferior thyroid artery. Using dyestaining method we confirmed that the end of the catheter was definitely in truncus thyreocervicalis and not in subclavian artery. Thereafter, the catheter was fixed. It was then lead to the outside of the skin on the shoulder. Other arteries except ascending colli artery, which supplies partially the spiral cord, were ligated in expectation of effective arterial injection through inferior thyroid artery. Two modes of the treatment consisting of 5-FU arterial injection and irradiation were combined. The total dosage of the anti-cancer drug was 3, 125mg and irradiation dose was 2, 200rads. The tumor in the posterior commissure disappeared, but it remained in the farther anterior part of the commissure. This suggests that inferior thyroid artery might supply blood only to the region of posterior crico-arytenoid muscle, namely the region of the posterior commissure. Therefore, we directly injected 5-FU into the cricothyroid branch artery using Thermo 27G needle under microscope and simultaneously employed the radiotherapy. This arterial injection method is useful for the treatment of the laryngeal cancer without giving any dysfunction of cervical organs. The arterial injection method should be further developed for the treatment of head and neck cancer.

Fluorometric Scanning of Fluorescamine-Labeled Peptides in Polyacrylamide Gels

Abstract A rapid and sensitive fluorescence technique has been described for detecting and quantitatively assaying peptides in poly-acrylamide gels. Peptides were labeled with fluorescamine, subjected to disk electrophoresis and scanned for fluorescence with a modified Gilford 2410-S linear transport. The fluorescence signal was linear with peptide concentration when electrophoresis was performed in a SDS system. Fluorescence gel scanning with fluorescamine is recommended as an important criteria of purity because of its great sensitivity and because it will reveal peptide contamination not detected with the conventional dye-staining method.

Starch gelation as a function of water content

SummaryA dye staining method has been used to determine the proportion of starch granules which are damaged as a function of initial water content during (a) microwave heating and (b) baking in a forced‐air‐convection oven.Although the granules are damaged much more rapidly on microwave heating, it was found that the proportion of damaged granules depends on the amount of water in the sample rather than on the method of heating. Samples of potato starch containing up to 30% water suffer no measurable damage. Beyond this point, damage increases with increasing moisture content, and all the granules are damaged provided the samples contain initially 55% or more water.

GENETIC CONTROL OF ERYTHROCYTIC ESTERASE FORMS IN MUS MUSCULUS

TARCH or agar gel electrophoresis coupled with azo dye staining methods has permitted the detection of genetically determined variants of several forms of nonspecific carboxylic esterases in a protozoan (ALLEN 1961), maize ( SCHWARTZ 1960, 1964), Drosophila (WRIGHT 1963; WRIGHT and MACINTYRE 1963), and house flies (VELTHUIS and VAN ASPEREN 1963). In mammals, inherited electrophoretic variants of esterases have been reported from the serum (POPP and POPP 1962; PETRAS 1963) and kidney (RUDDLE and RODERICK 1965) of the house mouse ( M u s musculus), and from red cell lysates of the deer mouse (Peromyscus maniculatus) (RANDERSON 1964) and of man (TASHIAN 1965). The present report describes the genetic control, and preliminary chemical characterization, of some electrophoretic forms of nonspecific esterases in hemolysates of the house mouse.