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PubMed1994

Silver-staining of proteins in polyacrylamide gels: a general overview.

On the basis of the physico-chemical principles underlying silver-staining of proteins, which are recalled in this paper, several methods of silver-staining of proteins after SDS electrophoresis in polyacrylamide gels or isoelectric focusing were tested. The most valuable protocols are presented in this report, including standard methods for unsupported gels and new methods devised for thin (0.5 mm) supported gels for SDS electrophoresis or isoelectric focusing and for staining of small peptides. Generally speaking, the most rapid methods were found to be less sensitive and less reproducible than more time-consuming ones. Among the long methods, those using silver-diammine complex gave the most uniform sensitivity. They require however special home-made gels and cannot be applied to several electrophoretic systems (e.g. systems using tricine or bicine as the trailing ion, or isoelectric focusing in immobilized pH gradients). For these reasons, protocols based on silver nitrate are of a more general use and might be favored. Future trends for silver-staining will also be discussed.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Silver staining method for DNA in polyacrylamide gels using eriochrome black T as a silver-ion sensitizer.

A sensitive silver staining method using eriochrome black T as a silver-ion sensitizer for DNA detection in polyacrylamide gels was developed. The sensitivity of this staining method was significantly improved by the new silver-ion sensitizer containing a diazo group, which has reducing power. The staining method lasted a total of approximately 15 min following a fixing step for 2 x 20 min. The detection limit of this staining method was 1-4 pg for PhiX174 DNA/HaeIII in both nondenaturing and denaturing polyacrylamide gels. This staining method was especially effective in low-base pair DNA, with a sensitivity that was approximately ten-fold higher than previously published silver staining methods.

Acrylic Resins↗
PubMed1985

Silver staining for selective detection of histones in polyacrylamide gels.

A simple silver-staining technique was developed for selective visualization of histones in polyacrylamide gels. The specificity of the stain was confirmed using a variety of protein mixtures and isolated histones. The staining procedure requires a relatively short time to perform (2.5-3 h), and the sensitivity to lysine-rich histones is comparable to that of the conventional Coomassie blue stain (about 0.1 microgram per band). A possible mechanism for the selective staining was deduced from a comparison with the widely used ultrasensitive silver staining.

Chemical Phenomena↗
PubMed1991

Meiotic chromosome structure. Kinetochores and chromatid cores in standard and B chromosomes of Arcyptera fusca (Orthoptera) revealed by silver staining.

The behaviour of two chromosome structures in silver-stained chromosomes was analyzed through the first meiotic division in spermatocytes of the acridoid species Arcyptera fusca. Results showed that at diakinesis kinetochores and chromatid cores are individualized while they associate in bivalents of metaphase I; only kinetochores and distal core spots associate in the sex chromosome. Metaphase I is characterized by morphological and localization changes of both kinetochores and cores which define the onset of anaphase I. These changes analyzed in both autosomes and in the sex chromosome allow us to distinguish among three different substages in metaphase I spermatocytes. B chromosomes may be present as univalents, bivalents, or trivalents. Metaphase I B univalents are characterized by separated cores except at their distal ends and individualized and flat sister kinetochores. At anaphase I sister kinetochores of lagging B chromatids remain connected through a silver-stained strand. The behaviour of cores and kinetochores of B bivalents is identical with that found in the autosomal bivalents. The differences in the morphology of kinetochores of every chromosome shown by B trivalents at metaphase I may be related to the balanced forces acting on the multivalent. The results show dramatic changes in chromosome organization of bivalents during metaphase I. These changes suggest that chromatid cores are not involved in the maintenance of bivalents. Moreover, the changes in morphology of kinetochores are independent of the stage of meiosis but correlate with the kind of division (amphitelic-syntelic) that chromosomes undergo.

Animals↗
PubMed1988

Silver staining for carboxymethylated metallothioneins in polyacrylamide gels.

A sensitive method for detecting metallothioneins (MTs) by use of silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of carboxymethylated MTs was developed. Carboxymethylation of metallothioneins is indispensable because it prevents their aggregation, thereby allowing each of them to be detected as a single band by SDS-PAGE. However, when the gel was subjected to the silver-staining method of C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert [(1981) Science 211, 1437-1438], the image of carboxymethylated purified MTs was totally negative. Pretreatment of the gel with 1% sodium thiosulfate just prior to the silver-staining procedure successfully reversed the negative image of carboxymethylated MTs. Further, they could be detected with a limit of nanogram levels per lane. This method can be applied to MTs in cell extracts from cultured cell lines treated with cadmium or to those from liver of cadmium-intoxicated mice.

Animals↗
PubMed1983

Silver stain for detecting 10-femtogram quantities of protein after polyacrylamide gel electrophoresis.

A rapid and highly sensitive silver stain and color stain were developed for visualizing proteins. The procedure is simple and the bands were clear. This silver stain detects 100 pg quantities of proteins. In order to stain quickly, sensitively, and sharply a protein matrix in a gel, the repeated shrinkage and swelling gel was developed with a hyper- and hypotonic solution to remove the sodium dodecyl sulfate (SDS) from SDS-protein complex and to generate influx of staining solution into the gel. We have found that the silver staining method with the repeated exposure to hyper- and hypotonic solution and a narrow well produced 10 fg order of proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1985

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections.

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

Acrylic Resins↗
PubMed1992

Nucleolar silver staining patterns of lymphocytes in sarcoidosis.

Bronchoalveolar lavage lymphocytes from 15 patients with pulmonary sarcoidosis and 8 healthy controls were investigated for nucleolar silver staining patterns and lymphocyte subpopulations. Patients with sarcoidosis had increased numbers of silver stained dots versus controls (2.20 +/- 0.24 versus 1.78 +/- 0.07; p less than 0.001). The number of silver stained dots showed the strongest positive correlation to helper cells (OKT 4+) (r = 0.781; p less than 0.0001). These results may be interpreted as further evidence of lymphocytic activation, especially of helper cells (OKT 4+) in pulmonary sarcoidosis.

Adult↗
PubMed1992

Silver staining of collagen type I after sodium dodecylsulphate polyacrylamide gel electrophoresis: effect of Maillard reaction.

Differences in the acidic silver staining, after sodium dodecylsulphate polyacrylamide gel electrophoresis, between the alpha 1 and alpha 2 collagen chains, as well as between rat-tail tendon and calf-skin collagen type I, were observed. The staining conditions at which the staining differences are both most expressed and reproducible were characterized. Age differences between staining of the alpha 1 CB6 fragment from young rats (2 and 12 months) and old rats (29 months) indicated that different susceptibilities of collagen species to the silver staining can be the result of different extents of some age-dependent post-translational modification, such as glycation. In vitro incubation of acid-soluble rat-tail tendon collagen with various sugars led to an increase in silver staining compared with samples incubated in the absence of sugar. This effect was inhibited by sodium cyanoborohydride, diethylenetriamine pentaacetic acid and aminoguanidine, i.e. compounds inhibiting the Maillard reaction at various stages. It can be concluded that the enhanced silver susceptibility of glycated collagen is related to advanced-phase Maillard reaction products attached to collagen.

Animals↗
PubMed1991

Cyclic appearance of cytoplasmic NOR-silver-stained particles in sea urchin embryos.

When sea urchin embryos were subjected to nucleolar organizer region (NOR)-silver staining, densely stained particles were observed in the cytoplasm. The appearance of these cytoplasmic particles (CPs) was cell-cycle dependent. During early development, the CPs were detected at interphase, but not during mitosis; they disappeared at metaphase and reappeared at telophase. The CPs appeared periodically even when embryos were treated with cytochalasin B or aphidicolin, which inhibits the progression of cytokinesis and nuclear division, respectively. By contrast, CPs were not detected in the colchicine-treated embryos in which both cytokinesis and nuclear divisions were prevented. The CPs were observed only in the embryos whose stage was early blastula (about 6th to 7th cleavage) or earlier; no CPs were detected even at interphase in the embryos at late blastula (about 8th to 9th cleavage) or later. Electron microscopic evaluation showed CPs to be granular structures, similar to heavy bodies. Also, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) showed that 95-kDa and 38-kDa proteins were the NOR-silver-staining proteins in sea urchin embryos. These proteins existed during the course of the cell cycles. These results suggest that (1) the cyclic appearance of the CPs or heavy bodies is closely related to the cell cycle as well as the programming of the embryogenesis, but independent of the cycle of cytokinesis and nuclear division; (2) 95-kDa and 38-kDa proteins are the major NOR-silver-staining proteins in sea urchin embryos.

Animals↗
PubMed1984

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes.

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.

Cell Cycle↗
PubMed1996

[Detection of point mutation of p53 gene by silver staining PCR-SSCP in paraffin-embedded malignant fibrous histiocytoma].

Silver staining PCR-SSCP method was used to detect point mutation of p53 gene in paraffin-embedded malignant fibrous histiocytoma (MFH) tissues. The abnormal shifting of the single-stranded DNA (ssDNA) was identified in 9 out of 16 cases. The positive figure of SSCP was 1,4,4, 3 in exon 5, 6, 7, 8, respectively. The mutant p53 protein was detected by microwave oven treatment and ABC immunohistochemistry. Positive nuclear staining was observed in 10 cases. The positive coincidence rate was 90.0% between SSCP and p53 protein expression. The mutation of p53 gene was not correlated with the subtypes of MFH. Our results indicate that detection of point mutations with silver staining PCR-SSCP is convenient, rapid and reliable in the screening of point mutation of genes.

Genes, p53↗
PubMed1995

The influence of proteins on silver staining of nucleic acids following polyacrylamide gel electrophoresis.

The influence, on silver staining, of proteins and restriction enzymes in polymerase chain reaction (PCR) products was studied in 12-20% polyacrylamide gels. For small DNA fragments (74, 41 and 33 bp) best results were achieved using 20% polyacrylamide gels. In 12% gels, restriction enzymes and also proteins used for PCR (bovine serum albumin, Taq-DNA polymerase) interfere with silver staining of nucleic acids.

DNA↗
PubMed2001

A silver staining method for single-cell gel assay.

The single-cell gel assay (comet assay) is a very useful microelectrophoretic technique for evaluation of DNA damage and repair in individual cells. Usually, the comets are visualized and evaluated with fluorescent DNA stains. This staining requires specific equipment (e.g., a high-quality fluorescence microscope), the slides must be analyzed immediately, and they cannot be stored for long periods of time. Here we describe, using human lymphocytes, some modifications of the silver staining for comets that significantly increase the sensitivity/reproducibility of the assay. This silver staining was compared with fluorescence staining and commercial silver stains. (J Histochem Cytochem 49:1183-1186, 2001)

Antineoplastic Agents↗
PubMed1994

Rapid silver staining and recovery of PCR products separated on polyacrylamide gels.

A rapid silver-staining procedure for DNA fragments in polyacrylamide gels is described. The time required for band detection is 15 min and the limit of sensitivity 3 pg/mm2. PCR products subjected to this rapid staining protocol are readily recovered from the gel by excision and elution by incubation at 95 degrees C for 20 min. Bands of up to 3 kb have been recovered and reamplified from either recently prepared or dried gels. The rapid staining protocol significantly decreases the processing time required for silver-stained polyacrylamide gels, which is of particular importance in diagnostic situations. The recovery protocol allows individual bands from complex mixtures to be easily recovered for sequencing or probe preparation.

Animals↗
PubMed1982

Silver staining of the nucleolus organizer regions (NOR) requires clusters of sulfhydryl groups.

Silver stainability of nucleolus organizer regions (NORs) appears to be correlated with the presence of grouped sulfhydryl (SH) side chains of proteins. In fact, heavy metals with high affinity for SH groups, such as Hg and Cu, do prevent the silver staining reaction. Ferricyanide, which is known to oxidize SH to disulfides, also prevents any further reaction with silver. On the other hand, alkali and reducing agents (mercaptoethanol, cyanide) do not affect silver stainability of the NORs. These results show that the silver staining reaction is not related to disulfide or persulfide groups and that alkali-soluble, acidic nuclear proteins per se do not play a major role in this process.

Cell Nucleolus↗
PubMed1991

Silver-stained structures in prostatic carcinoma: evaluation of diagnostic and prognostic relevance by automated image analysis.

The comparison of the diagnostic and prognostic significance of histology, immunohistochemical parameters (PSA, PSP), and silver-stained nucleolar organizer regions (AgNORs) was estimated in paraffin sections taken of 63 prostatic carcinomas prior to therapy. AgNORs were visualized with a one-step silver staining technique with the appropiate staining time determined by preliminary staining-time series. The mean AgNOR number per cell (n) and the mean AgNOR area per silver-stained dot (A) were determined by means of an automatic image analysis system. Thereby prostatic carcinomas exhibited multiple small AgNORs within their nuclei (n = 4.7, A = 0.09 micron 2), whereas benign prostatic epithelium showed few but large silver-stained particles (n = 1.8, A = 0.27 micron 2; p less than 0.001). This relationship was then calculated as a quotient of AgNOR number and area (NQ = n/A) which provided additional information for the diagnosis of malignancy as well as survival. Univariate survival analysis disclosed a set of four variables predicting death from prostatic cancer; cribriform growth pattern, AgNOR quotient, histological grade, and PSA immunoreactivity. Of these parameters, immunoreactivity of PSA failed to prove its prognostic significance in multivariate survival analysis (Cox model). No relation to prognosis was found for the number as well as the area of AgNORs alone. Therefore, image analysis proved to be a prerequisit for the feasibility of this promising technique by providing objective and reproducible results.

Acid Phosphatase↗