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A modification to a rapid hot methenamine silver stain is described that shortens the silver staining step of the procedure to one to five minutes. It also eliminates the requirement to manipulate hot solutions.
Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands.
A protein determination procedure which involves the densitometry of silver stained polyacrylamide gels is described. It involves calibration with bovine serum albumin and molecular weight markers on the same gel with the protein to be quantitated. The procedure is simple, rapid, reproducible and accurate and is more sensitive than other procedures for protein determination. The procedure is particularly useful in quantitating proteins purified in small amounts since the determination can be performed on the same gel used to check the purification. It avoids interference by detergents and other substances usually present in solutions of purified proteins. The procedure has been applied to the quantitation of a recently identified protein, aquaporin (CHIP28), assumed to be a major water channel in the red blood cell membrane. A quantitative analysis of a purified fraction of this protein shows that the 28 kDa component represents approximately two thirds of the protein content of the sample, with the remainder comprising a glycosylated, high molecular mass component. The procedure may be useful for quantitating proteins revealed on silver stained gels and could be included as a standard part of any protocol for protein purification.
Protein spots in silver-stained two dimensional gels were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including i) In-gel reduction, alkylation and enzymatic digestion ii) Extraction and desalting by using a pipette tip containing a small C18 micro-column (ZipTip(TM)) iii) Direct MALDI-TOF mass analysis and protein database searching. The results demonstrate that single silver-stained protein spots in a 2-DE-gel could be identified rapidly by this procedure and the use of the ZipTip(TM) pipette tip could increase evidently the sensitivity of the MALDI-TOF analysis. By using this the procedure, 10 protein spots in a silver-stained gel of 2-DE of crude venom of the spider S.Huwena were analyzed and identified.
Lack of silver staining of a variant chromosome 17 and its failure of involvement in satellite association indicate that this variant is a heteromorphism rather than a translocation of satellited material from any other acrocentric chromosome.
Lymph node biopsy specimens from 150 cases of reactive follicular hyperplasia, 52 cases of Hodgkin's disease and 30 cases of chronic lymphadenitis were studied. Clinical follow-up of cases for a period of two years at least was also considered. Formalin fixed and paraffin embedded tissues were stained with hematoxylin and eosin, and ammoniacal silver. Cytoplasmic accumulations of basic proteins were evidence in 30 out of 150 cases, but only 27 of these cases presented new lymph nodes with histological setting of Hodgkin's disease. The data suggest possible relationship between reactive follicular hyperplasia and Hodgkin's disease. However this relationship is not absolute as documented by two cases that being negative for the ammoniacal silver staining, were later diagnosed as Hodgkin's disease. Different types of atypical cells occurring in lymph nodes of reactive follicular hyperplasia were also studied. The potential use of ammoniacal silver staining in the early diagnosis of Hodgkin's disease is discussed.
A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).
Silver staining was used to estimate the expression of nucleolar organizing regions (NORs), and in-situ hybridization (ISH) with rDNA probes was used to estimate the relative content of rDNA in each NOR in chromosome preparations of the dormouse, Eliomys quercinus, a species with two NOR-bearing chromosome pairs. Both types of signals were sequentially investigated on every NOR by using an Ag-ISH sequential staining method, which made it possible to demonstrate that the relative amount of rDNA in a NOR in comparison with the other chromosomes of the complement determines its level of expression and its likelihood of becoming active, regardless of whether the NORs are homologous or not. We suggest that the NORs in each cell are activated in accordance with an established hierarchy. We propose a two-stage model to relate NOR structure and function, which is consistent with these results and with current knowledge on the molecular regulation of NOR transcription.
Silver staining (Howell and Black, 1980) was used in light and electron microscopic studies for detecting the localization of argentophilic nuclear proteins in fertilized ova and cleaving mouse embryos. No silver-stained nucleolus organizing regions (NORs) (Ag-NORs) were visualized in the metaphase chromosomes of the first cleavage mitosis. From the 2-cell stage on, metaphase chromosomes contained Ag-NORs. Argentophilic proteins were detected in the pronuclei of the 1-cell embryos, i.e. before transcription of the ribosomal genes started. After fertilization these proteins accumulated on the decondensing sperm chromatin and telophase maternal chromosomes, then migrated into the pronuclei to be stored in pronucleoli, and, during mitosis, were transferred into the cytoplasm. In the metaphase chromosomes of the cleaving embryos Ag-NORs adequately reflected the transcriptional activity of the ribosomal genes, whereas in pronuclei of the 1-cell stage embryos argentophilic proteins were not involved in this process, but are likely to play a part in the formation and maturation of pronucleoli, and in the cell cycle regulation.
Silver staining of nucleolar organizing regions (NOR) is common, but a standard protocol is lacking. A modification of a rapid silver nitrate staining technique for NORs is presented here. Advantages of the modified technique include reliability, speed, cost and the fact that it can be carried out in the light.
Silver-stained nucleolar organizer regions (AgNORs) have been widely used as a marker of cellular activity and proliferation. In a retrospective study, we investigated the potential value of AgNORs in 12 hypertrophic and 24 keloid scar tissues. Ten normal skin tissues served as controls. A standard silver-staining method was used, and the mean AgNOR count of dermal fibroblastic cells in each tissue was determined. In normal skin, the mean AgNOR count of dermal fibroblasts was 1.79+/-0.55, whereas fibroblastic cells in hypertrophic and keloid scars had mean AgNOR counts of 3.18+/-0.56 and 5.10+/-0.97, respectively. There was a statistically significant difference between the mean AgNOR counts of fibroblastic cells from normal skin, hypertrophic scar, and keloid scar [one-factor analysis of variance (ANOVA), p < 0.0001]. Our findings suggest that AgNOR count may be a useful marker for assessment of fibroblastic cell activity in hypertrophic and keloid scars, which may have potential value for histologic and biologic characterization of the two lesions.
The value of a one-stage silver staining technique for nucleolar organizer region (NOR)-associated protein was investigated as a predictor of clinical outcome in 100 rectal adenocarcinomas. In formalin-fixed tissues, a variety of silver-stained structures ranging from 0.5 to 7 microns are seen, the abundance of which bears no relation to prognosis, cell proliferation, or ploidy. Evidence is presented that the silver-stained structures are the result of coalescence of smaller particles caused by formalin fixation, and that assessment of NOR activity is not reliable in routinely formalin-fixed archival tissues.
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.
A sodium dodecyl sulfate-discontinuous polyacrylamide gel electrophoresis system for separation and quantitation of low-molecular-weight (75 to 10K Da) proteins from single muscle fibers is described. Slab gels (0.75 mm thick) were stained using an improved silver-stain technique which does not require photographic fixers in order to achieve low-level background staining. The modified staining procedure uses continuous flow washing to minimize the handling of gels. The procedure has high sensitivity and gave a linear response between approximately 2 and 70 ng of protein per band. In addition, a convenient method for mounting slab gels for photography, scanning, and long-term storage has been developed.
Nucleolar organizer regions (NORs) can be stained by a simple one-step silver technique; the black dots formed are termed AgNORs. Often AgNORs are tightly clustered, appearing as one silver-stained nucleolus (AgNu). We have assessed this technique as a possible prognostic indicator for thick (greater than 3.0 mm) primary cutaneous malignant melanoma (CMM). Three groups were studied: (A) seven thick CMM that had not metastasized 8-20 years after excision; (B) three thick CMM that developed metastases 6-9 years after excision; and (C) twelve CMM that presented with metastases or developed them within 4 years of excision. Two methods of counting silver-stained black dots in nuclei were employed: one method counted easily discernible black dots consisting of AgNus and dispersed AgNORs; the other attempted to count actual AgNORs both dispersed and clustered within AgNus. Scores per nucleus by the first method were 1.5-6.7 in group A, 1.1-2.6 in group B, and 1.4-5.4 in group C. AgNOR counts by the second method were 6.2-13.0 in group A, 5.4-8.9 in group B, and 5.3-10.5 in group C. No significant difference was present between groups for scores by either method. Due to the subjectively, technical difficulty, non-reproducibility, and tedium associated with the second method of attempting to count individuals AgNORs, the first method is recommended. It is concluded that this technique is of no value in predicting prognosis for CMM.
Chromium (III) enhanced the sensitivities of diamine silver staining of four proteins between 6- and 50-fold over that of the Coomassie Brilliant Blue (CBB)-chromium modified thiosulfate-silver staining method (Zhou et al. Biotechnology Letters, 2002, 24: 1561-1567). Using six dsDNA fragments, the detection limits of this new method was 10 to 30 pg per band, being 10- to 25-fold more sensitive than previous methods.
A rapid and highly sensitive silver staining method, originally developed for the detection of proteins, was slightly modified to detect nucleic acids in polyacrylamide gels. The second exons of the histocompatibility antigen HLA-DQA 1 and DQB 1 genes were selectively amplified from genomic DNA by the polymerase chain reaction (PCR). Digestion of the PCR products by endonucleases, followed by their size-separation on polyacrylamide gels and visualization by silver staining, allowed us to define the HLA-DQ alleles of the genomic DNA. The intensity of staining of digested PCR-amplified DNA is linear from at least 8 to 18 ng for fragments of lengths ranging from approximately 40 to 200 bp. Thus, silver staining in combination with PCR and allele-specific restriction fragment length polymorphism provides a simple, safe, and rapid method for accurate definition of HLA-DQ alleles at the nucleotide level in the clinical typing laboratory.
Different quantitative parameters of nucleolar silver staining have been studied in the cambial rat trophoblast cells on the 12th, 13th and 14th days of gestation. It has been shown that the number of Ag-positive granules in the nucleoli varied from 10 to 120. The number and the total area of silver stained granules in the nuclei increased progressively in the course of polyploidization, but was not doubled passing to the next ploidy level. Nevertheless, nucleolar area increased proportionally to the ploidy degree. The mean number and the total area of Ag-stained granules as well as the nucleolar area estimated for each ploidy level did not change significantly in the course of placenta development, suggesting an unchanged level of NOR activity at the studied stages of trophoblast cell differentiation. The data obtained on the interphase nucleoli differ from the data of the analysis of the metaphase Ag-NOR at the same period of placenta development, suggesting a diversity in the interphase and metaphase NOR organization. A proportion of cells with different number of nucleoli in the cambial rat trophoblast cells was maintained unchanged in the studied period of the placenta development, the majority (80-90%) of cells contained from 1 to 3 nucleoli. Such a proportion was similar in the cells of different levels of ploidy up to 16c. In this connection the association of NORs is suggested to be in relation with switching from the polyploid mitotic cycle to the endoreduplication leading to polyteny.