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PubMed1993

Towards stoichiometric silver staining of proteins resolved in complex two-dimensional electrophoresis gels: real-time analysis of pattern development.

Toward the ultimate goal of deriving quantitative protein data from silver-stained proteins resolved in complex two-dimensional electrophoresis gels we have performed computerized real-time analysis during pattern protein "silver stain" development. Three points emerged from this study: (i) Development time is a far more important variable than originally envisioned. (ii) Previous studies which, on the basis of a single development time, sought to relate silver stain slope and threshold properties to amino acid composition are non-informative since it will be shown that slope values change markedly with development time. (iii) The combination of real-time analysis and total protein load consideration allows, for the first time, protein "spots" to be grouped into categories, which in turn aid in quantification.

Animals↗
PubMed1988

'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining.

The crosslinker diacrylyl-piperazine produces polyacrylamide gels which display improved electrophoretic separation of proteins and better physical strength. It also produces gels with improved detection of proteins by ammoniacal silver staining by reducing the background. This reduced background provided us with an opportunity to investigate residual background staining caused by the catalytic reagents utilized in the polymerization of acrylamide gels. The commonly used catalyst system, tetramethyl-ethylenediamine and ammonium persulfate was shown to be responsible for the yellow staining background found after a prolonged development time with silver staining. An alternate catalyst system has been designed to decrease further the formation of this background staining. Dimethyl-piperazine or tetramethylethylenediamine, potassium or ammonium persulfate, and sodium thiosulfate are shown to provide for gels which have excellent mechanical and staining characteristics. These catalytic systems produce little background staining despite prolonged development time with the ammoniacal silver stain, and they reduce background staining with the dichromate silver stain.

Acrylic Resins↗
PubMed1977

Visualization of ribosomal gene activity: silver stains proteins associated with rRNA transcribed from oocyte chromosomes.

Cricket oocyte chromosomes were stained with silver at pachytene when certain chromosome regions are active in rDNA amplification and rRNA transcription. The silver preferentially stained the known locations of 18S + 28S ribosomal cistrons. Cytochemical tests revealed that the silver binds neither to the rDNA nor transcribed rRNA, but rather to proteins which rapidly associate with the freshly-transcribed rRNA. As rRNA transcription proceeds, the quantity of silver stainable proteins progressively increases. The silver procedure can be used to visualize gene activity at the rDNA sites with conventional light microscopy.

Animals↗
PubMed1987

Ultracytochemical detection of heteropolysaccharides in the cat submandibular gland.

We have investigated the morphology and the ultrastructural distribution of complex carbohydrates in the cat submandibular gland using the high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP), the low iron diamine-thiocarbohydrazide-silver proteinate (LID-TCH-SP), and the periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequences. The results of morphological analyses show that, in addition to already known cellular populations, in striated ducts a further cellular type containing glycogen is present. The histochemical staining showed that demilunar cells secrete polysaccharides that have carboxylated but not sulphated glycosidic residues, whereas acinar cells elaborate secretion products rich in carboxylated and sulphate-containing complex carbohydrates. Vicinal glycol groups are present as constituents of the secretion in demilunar, acinar, and ductal cells.

Animals↗
PubMed1993

A dual staining method for neutral carbohydrates and DNA (deoxyribonucleic acid) using periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) and Feulgen (Hot HCl-Schiff) techniques.

A dual staining method was established for the histochemical detection of neutral carbohydrates and deoxyribonucleic acid (DNA) in light microscopy. The method consisted of combined periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) and hot hydrochloric acid (H-HCl)-Schiff (Feulgen) procedures, which produced reaction products of blackish and magenta shades respectively. The present dual staining method is believed to be unusually useful for the light microscopic histochemical studies on neutral carbohydrate-containing cells, since it exhibits a high contrast between these two shades and reveals distinctly the localization of cytoplasmic neutral carbohydrates and nuclear DNA in one and the same tissue section.

Animals↗
PubMed2006

Formation of epsilon-formyllysine on silver-stained proteins: implications for assignment of isobaric dimethylation sites by tandem mass spectrometry.

Considerable effort is focused presently on the detection and comprehensive assignment of post-translational modifications of proteins. Obviously attention must be paid to the possibility of chemical modifications that may occur to protein samples during sample handling and manipulation prior to analysis by tandem mass spectrometry. This is of particular concern when a modification is isobaric with the mass differential in common with a known post-translational analog. Here we provide evidence that silver staining protocols that use formaldehyde can result in epsilon-formylation of lysine residues. This modification is in fact isobaric with the important product of methyltransferases, epsilon,epsilon-dimethyllysine. Without exercising proper caution the analysis of silver-stained protein samples by mass spectrometry looking for dimethylation of lysine will yield a significant number of misassigned sites of modification. High accuracy measurements of the mass of the precursor ions and their fragments are required to eliminate this uncertainty. The occurrence of dimethylation of the epsilon-amino function of lysine residues has been reported often in histones. For histone samples excised from silver-stained gels, we found that most sites initially assigned to be dimethylated by automatic search engines under standard search parameters (100 ppm error tolerance) are actually in fact formylated. Caution must be exercised when data obtained from instruments unable to perform high accuracy mass measurements (better than 5 ppm) are to be interpreted.

Amino Acid Sequence↗
PubMed1985

Stereoscopic back-scattered electron imaging of silver-stained proteins in nucleoli.

By using simultaneously the AgNOR silver staining method, back-scattered electron imaging mode and stereo-tilt in scanning electron microscopy (SEM), it is possible to observe the nucleus through the cell surface, the nucleolus, and the tri-dimensional distribution of the Ag-NOR-associated acidic proteins. In C3H10T1:2 cells and their 7-12-dimethylbenz-alpha-anthracene-treated transformants, the staining demonstrates several intranucleolar silver-staining granules (SSG), surrounded by a weakly staining region. The SSG may represent the fibrillar center (FC) and the weakly staining region, the fibrillar dense component (FD). This component can link several SSG together to form a "rope-like structure". In cells with no visible nucleolus and inactive nucleolar organizer regions (NORs) the silver-staining granules are less numerous, close together and the presumed fibrillar dense components are not visible. The SSG are located more peripherally, and the weakly staining region and the "rope-like structure" are less prominent in control cell nucleoli than in transformed cells with a comparatively high rate of RNA synthesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1975

Mental retardation, hypotonia, and generalized seizures associated with astrocytic "residual" bodies. An ultrastructural study.

Two siblings suffering since birth from convulsions, hypotonia, and mental retardation are presented. In the older sibling (eight and one-half years of age) frontal lobe biopsy revealed abnormal cytosomes with lamellar profiles in astrocytes, macrophages, and to a lesser degree in neurons. Similar cytosomes have not yet been reported in cases of sphingolipidoses or in late infantile-juvenile amaurotic idiocy. These cytosomes stained intensely with silver proteinate, an ultrastructural cytochemical stain for carbohydrate moieties. In contrast, lipofuscin did not stain with silver proteinate. Multilamellar (crescentic curvilinear) cytosomes from a reported case of late infantile amaurotic idocy (Batten-Vogt-Spielmeyer disease) did not stain with silver proteinate. Abnormal cytosomes were not found in blood cells, liver, and peripheral nerve. In the younger sibling (14 months old) postmortem ultrastructural studies of cerebral tissue showed very few abnormal cytosomes. On the basis of the clinical and ultrastructural findings, we conclude that these two cases can be distinguished from those with multilamellar (crescentic-curvilinear) inclusions and from cases of the so-called "neuronal ceroid-lipofuscinosis" syndrome.

Astrocytes↗
PubMedDate not supplied

Quantitation of human leukocyte proteins after silver staining: a study with two-dimensional electrophoresis.

The quantitative attributes of human leukocyte proteins detected by silver staining two-dimensional electrophoresis (2-DE) gels were studied by using computer-assisted data analysis. Experiments included (a) analysis of replicate patterns of the same sample, (b) analysis of different dilutions of the same sample, and (c) analysis of samples from different individuals. Over 200 proteins were observed to have coefficients of variation (CV) less than or equal to 15% when data from replicate patterns were analyzed. In contrast, 8 proteins had CV values of less than or equal to 15% when data from different samples were analyzed. The dilution experiment showed that a majority of the proteins detected with some consistency (i.e., observed in at least 80% of the patterns) have a linear relationship between the amount of protein loaded onto a 2-DE gel and the spot volume in the final 2-DE pattern. The slope of the curves and the deviation from linearity were found to be quite protein-specific. These results indicate that optimization of sample purity and minimization of staining protocol variables are required to limit the background quantitative variability between and within 2-DE runs to a level that will allow detection of quantitative changes indicative of biological responses.

Blood Platelets↗
PubMed1998

Effects of sex steroids on silver stained proteins of nucleolar organizer regions (Ag-NOR) in the rabbit uterus.

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

Animals↗
PubMed1994

The use of periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) procedure for the histochemical detection of neutral carbohydrates in the circumventricular organs of the rat.

A periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) procedure was applied to the rat circumventricular organs (CVOs), which are known to be specialized structures in the brain. In the rat CVOs, vivid PA-TCH-SP-PD reactions were obtained in the apical surface and cytoplasm of the ependymal cells of the subcommissural organ (SCO) and the epithelial cells in the choroid plexuses (CPs) examined, and similar positive reactions were detected in the vascular walls and perivascular connective tissues in all the CVOs tested. Further, varying intensities of PA-TCH-SP-PD reactions were noted in the neuronal and glial networks of the organum vasculosum laminae terminalis (OVLT), subfornical organ (SFO) and area postrema (AP). The results obtained in the present study indicate that in the rat CVOs the histologic structures mentioned contain varying amounts of neutral carbohydrates and possible histophysiological significances of these carbohydrates in these organs have been discussed with references to their functions.

Animals↗
PubMed1990

Silver staining of proteins and DNA.

Silver stains offer high sensitivity for the detection of proteins and DNA separated on gels and membranes. These stains depend on the reduction of ionic to metallic silver.

Ammonia↗
PubMed1981

Binding of glycosomes to endoplasmic reticulum and to intermediate filaments in cardiac conduction fibers.

Canine cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of cardiac conduction fibers were fixed with glutaraldehyde, postfixed with OsO4 and treated en bloc with uranyl acetate. Ultrathin sections were stained sequentially by periodic acid-thiosemicarbazide-silver proteinate, for glycogen, and by uranyl acetate and lead citrate, for protein. Glycosomes (organelles composed of glycogen and protein) were found attached to membranes of endoplasmic reticulum (ER) and to intermediate filaments (IF). The bound glycosomes retained their integrity after treatment of tissue en block with uranyl acetate, a procedure that destroyed free glycosomes in the same cell. the coincident distributions of ER and IF and their association with glycosomes suggests a functional relation between there three components.

Acids↗
PubMed2002

Ultrasonographic appearance of edema caused by injections in the mammary gland attachments of dairy cows.

OBJECTIVE: To determine the ultrasonographic appearance and detectability of edema induced by SC injection of mild silver protein suspension in the mammary gland attachments of dairy cows. DESIGN: Prospective study. ANIMALS: 6 lactating cows. PROCEDURE: In each cow, the number of quarters that received injections was randomly assigned. A mild silver protein susoension was injected SC into cranial and caudal mammary gland attachment sites. The number of injections and volume injected were determined on the basis of the appearance of the mammary gland and the desired subjective visual effect. Seventeen sites were chosen for injection and 7 sites did not receive injections. Ultrasonographic images were obtained 1 day prior and 6 days after injections were started. Cows received injections 1, 3, and 5 days after initial sonography. The sonographer was unaware of which sites received injections. RESULTS: Ultrasonography revealed alternating hypoechoic and hyperechoic bands at injection sites. Certain injections caused the intimal surface of the subcutaneous abdominal vein to develop a corrugated appearance. All injection sites were correctly identified ultrasonographically (100% sensitivity, 100% specificity) with a positive and negative predictive value of 1.0. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that mild silver protein suspension injected SC to enhance the appearance of the mammary glands of dairy cows can be readily detected by ultrasonography. Detection of injection sites should be made on the basis of the distribution and ultrasonographic appearance of edema.

Animals↗
PubMed1981

The appearance of carbohydrate-rich material in the developing Golgi apparatus of amoebae.

The silver proteinate reaction was used to stain carbohydrate-rich substances in normal Amoeba proteus and in the developing Golgi apparatus of renucleated amoebae. Normal cells contained stained material, which probably is glycoprotein, in the cell surface, cisternae at the concave pole of the Golgi apparatus, and cytoplasmic vesicles and vacuoles. Previous radioautographic studies had shown tht glycosylation occurs in the Golgi apparatus, and that material in the Golgi apparatus is precursor to the cell surface. Amoebae were enucleated for 5 d, which results in a decline of the Golgi apparatus, the disappearance of the glycoprotein-containing cisternae preceding that of the rest of the organelle. A new nucleus was then transplanted into the enucleate amoebae, bringing about the regeneration of the Golgi apparatus. small curved cisternae that appeared 30 min after renucleation lacked staining with silver proteinate. By 1 h after renucleation, however, the content of cisternae toward the concave poles of Golgi bodies stained with silver proteinate. The Golgi apparatus in cells fixed 6 h and 1 d after operation resembled that of normal amoebae in both morphology and staining pattern. The results suggest that the developing Golgi apparatus acquired the capacity to participate in assembly of cell-surface material within 1 h after renucleation. This occurred before development of the normal enzymic activity of the Golgi apparatus was completed.

Amoeba↗