Use Of Silver Staining Research Articles

Gel electrophoretic separation of proteins from cultured neuroendocrine tumor cell lines.

Neuroendocrine tumors (NET) often develop asymptomatically and are detected at a late stage. Currently, there exist certain markers of NET that occur only in the advanced stages of the disease. Still, there is need to develop markers specific of the early stage of cancer development. Nevertheless, biomarkers are mostly low‑abundant proteins and require separation from complex protein mixtures, which remains a major challenge. The goal of the present study was to optimize one‑dimensional‑polyacrylamide gel electrophoresis (1D‑PAGE) for separation and comparison of protein composition from neuroendocrine tumor samples. 1D‑PAGE was optimized by modification of the gel concentration and by comparison of different gel staining protocols. In addition, several steps prior to electrophoresis were carried out to purify and preliminarily reduce the complexity of the sample. The results of these optimization steps indicated that use of an albumin removal kit can considerably decrease the amount of albumin in the samples, thereby allowing to detect proteins of low abundance. Optimal separation of the sample was obtained using a 12% polyacrylamide gel. Furthermore, the use of silver staining allowed detection of proteins at nanogram levels, whereas for Coomassie Brilliant Blue staining, the detection limit was 10 times higher. Optimization of the sample preparation workflow and parameters of the electrophoretic separation allowed to reduce the complexity of the studied material and facilitated further identification of proteins of low abundance in the sample. This study demonstrated that analysis of the secreted proteome of NET cells by 1D‑PAGE is a simple and suitable tool for the identification of potential NET protein biomarkers.

What Is The New and Sensitive Tumor Marker for Detection of Different Kidney Tumors? Modern Study to Isolation, Purification and Characterization of N-Acetyl Galactosamine Binding Lectin From Sera Of Patients With Kidney Cancer

The present study was designed to investigate lectins in sera of patients with kidney tumors, in addition to non tumoral kidney disease patients. Fifty five patients of malignant kidney tumors were enrolled in addition to 23 patients of benign kidney tumors, and 18 patients of non tumoral kidney diseases used as control groups, in addition to 46 healthy individuals were also investigated. The age of patients and healthy individuals were 10-90 years. The measurement of total serum proteins revealed significant (p < 0.001) decrease in patients of malignant tumors when compared with those of benign, non tumoral diseases, and healthy individuals. The conditions of the hemagglutination assay of serum lectin activity were optimized. They were Tris buffer of 20 mM and pH 7.4, 60 mM CaCl2, 800 µg of defatted serum, 30 ˚C for serum samples, 60 minutes for serum samples, and human blood of group A+ suspension with 1.4 optical density. The measurement of the specific hemagglutination activity of lectins demonstrated significant (p < 0.001) elevation in patients of malignant tumors when compared with those of other patients and healthy individuals. Lectin activity was pointed out to be significantly (r = 0.767 at p < 0.0005) positively correlated with stage of malignancies. The cutoff value of the specific hemagglutination activity was found to equal 6 SHU for discriminatory malignant kidney tumors. Serum lectins activity were indicated to be inhibited by galactose, mannose, lactose, and N-acetyl galactosamine. Purification of lectin from sera of patients with malignant kidney tumors by affinity chromatography with the use of silver stain revealed N-Acetyl Galactosamine Binding Lectin (GalNAcBL). The purified folds and the yield was 178 with 32.4%. The polyacrylamide gel electrophoresis (PAGE) of purified lectin demonstrated one band consisted lectin activity. The approximate molecular weight of GalNAcBL was determined and found to be 63.83. Purified lectin was characterized through the assessment of the capability to agglutinate RBCs, inhibition by EDTA, pH dependency, thermal dependent, and carbohydrate contents. GalNAcBL were observed to be calcium dependence lectins (C-type). These results suggest that the diagnosis of the specific hemagglutination activity of lectin is promising biomarker for discrimination of malignant kidney tumor patients and the purified lectin could be introduced in the field of biomarkers.

Hyperchlorhidrosis Caused by Homozygous Mutation in CA12, Encoding Carbonic Anhydrase XII

Excessive chloride secretion in sweat (hyperchlorhidrosis), leading to a positive sweat test, is most commonly indicative of cystic fibrosis yet is found also in conjunction with various metabolic, endocrine, and dermatological disorders. There is conflicting evidence regarding the existence of autosomal-recessive hyperchlorhidrosis. We now describe a consanguineous Israeli Bedouin kindred with autosomal-recessive hyperchlohidrosis whose sole symptoms are visible salt precipitates after sweating, a preponderance to hyponatremic dehydration, and poor feeding and slow weight gain at infancy. Through genome-wide linkage analysis, we demonstrate that the phenotype is due to a homozygous mutation in CA12, encoding carbonic anhydrase XII. The mutant (c.427G>A [p.Glu143Lys]) protein showed 71% activity of the wild-type enzyme for catalyzing the CO₂ hydration to bicarbonate and H(+), and it bound the clinically used sulfonamide inhibitor acetazolamide with high affinity (K(I) of 10 nM). Unlike the wild-type enzyme, which is not inhibited by chloride, bromide, or iodide (K(I)s of 73-215 mM), the mutant is inhibited in the submicromolar range by these anions (K(I)s of 0.37-0.73 mM).

Visualizing Quantum Dots in Biological Samples Using Silver Staining

Quantum dot (QD) based contrast agents are currently being developed as probes for bioimaging and as vehicles for drug delivery. The ability to detect QDs, regardless of fluorescence brightness, in cells, tissues, and organs is imperative to their development. Traditional methods used to visualize the distribution of QDs in biological samples mainly rely on fluorescence imaging, which does not account for optically degenerate QDs as a result of oxidative quenching within the biological environment. Here, we demonstrate the use of silver staining for directly visualizing the distribution of QDs within biological samples under bright field microscopy. This strategy involves silver deposition onto the surface of QDs upon reduction by hydroquinone, effectively amplifying the size of QDs until visible for detection. The method can be used to detect non-fluorescent QDs and is fast, simple, and inexpensive.

Enhanced Multicolor Resonant-Optical-Assay with Direct Text Output

The effectiveness of a novel multicolor biochip boosted by reducing cluster repulsion and in-situ silver enhancement has been demonstrated by using anti-serum albumine antibodies conjugated to gold nanoparticles on a vacuum metalized plastic film coated with nano-resonance driving ceramic multilayers. A dense and smooth vacuum deposited SiO approximately 1.6 top layer (50 to 300 nm thick) on a flexible thermoplastic polymer poly-ethylene-terephthalate-chip functionalized via poly-ethylenimine monolayer coating and chemical cross-linking was employed to immobilize capture antibodies. Following capturing of the human serum albumin antigen from analyte solution, the multicolor chip reacts with anti HSA-gold nanoparticles binding them in a nanometric distance to the resonant mirror of the device-visible to the eye as faint coloring of the chip surface. Following silver enhancement, a strong metallic angle-dependent color via Resonance Enhanced Absorption is observed. In this study, silver staining has been used for the first time to boost and shift the color of nano-resonance enhanced optical bioassays. The use of silver staining increases and significantly modifies the intensity of the resonance color and was done in less than 5 minutes directly on the chip. This novel methodology will find broad application in Point-of-Care diagnostic devices via a color signal output designed as a written text with high contrast to replace standard lateral flow devices just showing lines or dots.

What Is The New and Sensitive Tumor Marker for Detection of Different Kidney Tumors? Modern Study to Isolation, Purification and Characterization of N-Acetyl Galactosamine Binding Lectin From Sera Of Patients With Kidney Cancer

The present study was designed to investigate lectins in sera of patients with kidney tumors, in addition to non tumoral kidney disease patients. Fifty five patients of malignant kidney tumors were enrolled in addition to 23 patients of benign kidney tumors, and 18 patients of non tumoral kidney diseases used as control groups, in addition to 46 healthy individuals were also investigated. The age of patients and healthy individuals were 10-90 years. The measurement of total serum proteins revealed significant (p < 0.001) decrease in patients of malignant tumors when compared with those of benign, non tumoral diseases, and healthy individuals. The conditions of the hemagglutination assay of serum lectin activity were optimized. They were Tris buffer of 20 mM and pH 7.4, 60 mM CaCl2, 800 µg of defatted serum, 30 ˚C for serum samples, 60 minutes for serum samples, and human blood of group A+ suspension with 1.4 optical density. The measurement of the specific hemagglutination activity of lectins demonstrated significant (p < 0.001) elevation in patients of malignant tumors when compared with those of other patients and healthy individuals. Lectin activity was pointed out to be significantly (r = 0.767 at p < 0.0005) positively correlated with stage of malignancies. The cutoff value of the specific hemagglutination activity was found to equal 6 SHU for discriminatory malignant kidney tumors. Serum lectins activity were indicated to be inhibited by galactose, mannose, lactose, and N-acetyl galactosamine. Purification of lectin from sera of patients with malignant kidney tumors by affinity chromatography with the use of silver stain revealed N-Acetyl Galactosamine Binding Lectin (GalNAcBL). The purified folds and the yield was 178 with 32.4%. The polyacrylamide gel electrophoresis (PAGE) of purified lectin demonstrated one band consisted lectin activity. The approximate molecular weight of GalNAcBL was determined and found to be 63.83. Purified lectin was characterized through the assessment of the capability to agglutinate RBCs, inhibition by EDTA, pH dependency, thermal dependent, and carbohydrate contents. GalNAcBL were observed to be calcium dependence lectins (C-type). These results suggest that the diagnosis of the specific hemagglutination activity of lectin is promising biomarker for discrimination of malignant kidney tumor patients and the purified lectin could be introduced in the field of biomarkers.

Enhanced Detection of Microsatellite Instability and Mismatch Repair Gene Expression in Cutaneous Squamous Cell Carcinomas

Microsatellite instability (MSI) is a phenotypic characteristic of tumors with biallelic inactivation of mismatch repair genes, such as MSH2 or MLH1, and contributes to malignant transformation. The aim of this study was to examine the prevalence of MSI in cutaneous squamous cell carcinoma (SCC) using a PCR and fluorescent-based detection system. These methods of analysis offer several advantages over the use of silver staining and autoradiographic techniques. We also aimed to determine if MSI status correlated with expression of the MSH2 and MLH1 mismatch repair proteins in these cutaneous SCC samples. The MSI status of 22 histologically confirmed invasive cutaneous SCC samples were analyzed at five microsatellite markers (the National Cancer Institute's Bethesda panel of two mononucleotide and three dinucleotide markers) using a PCR and fluorescent-based detection system. Immunohistochemical analysis of MSH2 and MLH1 protein expression was also carried out on the SCC samples. Only one case of cutaneous SCC displayed MSI. This was found at just one of five markers, and thus was low frequency MSI. All 22 cutaneous SCC cases strongly expressed MSH2 protein. Eighteen (82%) of the cutaneous SCC cases showed moderate to strong expression of MLH1 protein. The remaining four cases of cutaneous SCC were negative for MLH1 protein. Therefore, the majority of the SCC patients analyzed showed a correlation between absence of MSI and expression of MSH2 and MLH1 proteins. MSI is uncommon in cutaneous SCC. In addition, MSH2 was strongly expressed in all SCC samples analyzed and appeared to be upregulated when compared with the corresponding normal tissue. MLH1 protein was not detected in 4 of 22 SCC cases, although it was expressed in the corresponding normal tissue, suggesting that inactivation of MLH1 may be a late event in a subset of invasive SCC cases.

Sensitivity and variability of visual scoring in the comet assayResults of an inter-laboratory scoring exercise with the use of silver staining

Sensitivity and variability of visual scoring in the comet assayResults of an inter-laboratory scoring exercise with the use of silver staining

Sensitivity and variability of visual scoring in the comet assay: Results of an inter-laboratory scoring exercise with the use of silver staining

Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 °C to doses of 0, 5, 10, 25, 50, 100 and 200 μM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75‰. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 μM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 μM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method

Silver Staining of Nucleolar Organizer Regions

The use of silver staining to identify nucleolar organizer regions (NORs) has increased dramatically in recent years. NORs are cellular structures present during the mitotic and G1 phases of cell division. The number and size of silverstained NORs (AgNORs) have been associated with increased rates of proliferation and decreased nuclear stability. The clinical utility of the evaluation of AgNORs in the differential diagnosis and prognosis of cancer is currently being studied. Most histology laboratories are equipped to perform the staining procedure, which is both simple and inexpensive. Routine formalin fixation and staining times that do not exceed 30 min are satisfactory for most tissues. Although computer-based image cytometry generates objective analyses that differentiate subtle differences in the expression of AgNORs, the types of patterns of interest, the variation of expression between tissues, and the number of AgNORs to measure per case have not yet been established. This lack of standardization has led to contradictory data concerning the clinical utility of the evaluation of AgNORs. In non-Hodgkin's lymphoma, bladder cancer, malignant melanoma, and brain cancer, however, the number of AgNORs more often correlates with the prognosis of the patient than with staging of the lesions. In other cancers, including prostate cancer, a consistent correlation of the number of AgNORs with prognosis has not been shown. Future investigations using standardized measurement protocols and analyses of the size, number, and shape of AgNORs compared to molecular activity events in the nucleus may offer more significant test outcomes. From those findings, new measurement algorithms may be developed to more accurately measure and assess the usefulness of AgNORs in situ. (The J Histotechnol 19:241, 1996)

Use of silver staining to detect nucleic acids.

AbstractSilver stains are useful for the detection of nanogram amounts of proteins or nucleic acids in acrylamide gels or on various membranes. They have been shown to be more sensitive than organic stains in detecting proteins and DNA. They are capable of detecting as little as 0.03 ng/mm2 of DNA (1). In addition, silver stains avoid the mutagenic hazards presented by both ethidium bromide and radioactive detection methods.KeywordsSilver NitrateBackground StainingMetallic SilverSilver Nitrate SolutionDichromate OxidizerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Combined use of silver staining of the retrograde tracer WGAapoHRP-Au and pre-embedding immunocytochemistry for electron microscopy: demonstration of dopaminergic terminals in synaptic contact with striatal neurons projecting to the substantia nigra in the rat.

We investigated the applicability of the pre-embedding immunoperoxidase technique to WGAapoHRP-Au retrograde tracing. After injection of the tracer into the substantia nigra of rat, the brain was fixed and cryostat sections were immunostained for dopamine. The sections were osmicated and silver-stained to amplify the colloidal gold particles. Products of both the immunoperoxidase staining and the silver staining could be detected and distinguished by electron microscopy at low magnification. The ultrastructure was so well preserved that synaptic characteristics could be investigated. Dopaminergic terminals were demonstrated to synapse with striatal neurons projecting to the substantia nigra.

Cystic fibrosis proteins detected by electrophoretic techniques.

For the detection of the cystic fibrosis protein (CFP) in serum of cystic fibrosis (CF) carriers, thin-layer polyacrylamide gel isoelectric focusing proved inappropriate as a diagnostic test, but was useful for screening fractions on purification of CFP by chromatofocusing on a Mono P column. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis an Mr 12,000 protein (P12) was found in most CFP-positive sera, indicating good correlation between these two CF-associated proteins. Detection of the P12 protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was well reproducible and less delicate than IEF. The technique was also used to purify P12 from serum by two successive preparative electrophoresis steps in a 7.5-15% gradient and 15% homogeneous gel. The use of silver staining revealed that P12, which was present in all sera of CF patients and carriers with variable intensities, was also present in trace amounts in normal sera.

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.

Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.

Use of silver staining (Dieterle's stain) in the diagnosis of cat scratch disease

Lymph node involvement in cat scratch disease is characterized by a granulomatous lymphadenitis with micro-abscesses. Recently, it has been shown that the possible aetiological agent is a micro-organism which stains with some silver stains. In this study 60 lymph nodes were studied from 60 patients, using a modified Dieterlé's stain. In 40 cases (66%) rod-shaped bacilli with rounded extremities were observed. They were negative both with the Gram and Ziehl-Neelsen stains. Since such micro-organisms were demonstrated in the majority of the nodes examined, it can be concluded that silver stains are appropriate for the diagnosis of this condition.

The use of silver stain in sodium dodecyl sulfate polyacrylamide gel electrophoresis of immunoprecipitates obtained by crossed immunoelectrophoresis and liquid precipitation

AbstractSilver staining was examined as a means for detecting antigenic polypeptides separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) after recovery from immunoprecipitates produced by crossed immunoelectrophoresis and liquid precipitation. Both precipitin procedures resulted in detection of the expected polypeptides with bovine serum albumin (BSA) and human transferrin as model antigens, as well as microheterogeneity of BSA, whereas antigenic polypeptides were rarely detected with Coomassie Brilliant Blue staining of SDS‐PAGE. The technique is advantageous over the use of radiolabeled antigens in being less expensive and time consuming and over antibody‐blot procedures in detecting antigenic activity prior to the denaturing conditions of SDS‐PAGE. Possible drawbacks are the appearance of antibody polypeptide chains on the electrophorograms as well as other non‐antigenic components when crude extracts of virally‐infected cells are used as antigen.

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 m urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.

Unstable behavior of the chromosome satellite combined with an elongated connecting thread during mitosis inBrodiaea uniflora engl.

A preliminary examination by use of silver staining revealed polymorphism with respect to the satellite and the connecting thread of the secondary constriction in the mitotic metaphases ofBrodiaea uniflora. Detailed studies on the supernumerary chromosome fragments (SCFs) were carried out in the bulbs which had only a single satellited chromosomes in their chromosome complements. The SCFs never took part in equatorial plate formation at metaphase and usually lagged in the middle between the daughter chromosome complements at anaphase. They could be discriminated into three categories: (1) a compact fragment consisting of segments that stained deeply and faintly with Giemsa, (2) a dumbbell-shaped fragment, and (3) a dual-structured fragment which lacked faintlystained segments. The faintly-stained segment and the thin thread of the dumbbell-shaped SCF were specifically stained black with silver. At metaphase the satellites combined with extremely elongated connecting threads were perpendicular to the equatorial plates. Three types of bridges by the connecting threads were seen at anaphase: (1) a bridge with a satellite in the middle, (2) a bridge with a satellite in a pole, and (3) a bridge without a satellite. Breakage frequently occurred on these threads. It is concluded that the SCFs detected in this plant originate from the satellites through breakage of the connecting thread at anaphase, and the connecting threads are subject to suffering the breakage-fusion-bridge cycle.

Microscale multisample two-dimensional electrophoresis of proteins in human serum, cerebrospinal fluid, and urine.

In this technique, in which no denaturing agent is used, proteins in human serum, cerebrospinal fluid, and urine are separated by isoelectric focusing in cylindrical 40 g/L polyacrylamide gels of capillary size (1.3 x 35 mm) for 40 min, followed by electrophoresis in 40--170 g/L polyacrylamide linear gradient gel, with use of 38 x 35 x 1 mm slab gel, for 1 h. Only 2 microL of untreated human serum is required to obtain clear protein-distribution patterns, made visible by Coomassie Blue staining. By use of silver staining, proteins in unconcentrated cerebrospinal fluid can be made visible. An apparatus we devised for microscale two-dimensional electrophoresis enables us to analyze eight protein samples simultaneously.