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PubMedDate not supplied

[Isoelectric focusing with silver impregnation of proteins].

Silver impregnation of polyacrylamide gel sheets after isoelectrofocusing is described. This method of staining allows to increase the sensitivity of the traditional isoelectrofocusing 5 to 10 times, as compared with staining of gels by Coumassi brilliant-blue R-250 (in case of analysis of complex protein mixtures). When analyzing individual protein preparations, the sensitivity of isoelectrofocusing with silver staining is within the range of several dozens of ng per sample. A special attention is paid to methods permitting to decrease the non-specific background staining of gel by silver.

Amphibians↗
PubMed1977

Cytochemical duality of neurosecretory material in the hypothalamo-posthypophysial system of the rat as related to hormonal content.

Cytochemical methods using silver proteinate, silver methenamine an potassium ferrocyanide + OsO4 for ultrastructural detection of glycoproteins allow, in the posthypophysis and the magnocellular nuclei of the rat, differentiation of two types of fibres and neurons: one type containing negative granules with a homogenous content of low electron density, the second type containing granules which demonstrate a ring shaped deposit either of silver or of potassium ferrocyanide-osmium complex, likely to be related to a glycoprotein component. The difference between these two types is increased by prestaining "en bloc" with uranyl acetate before the silver proteinate reaction. A similar investigation was carried out on the vasopressin deficient Brattleboro rat; the neurosecretory material, present in some endings and neurons only, is of the nonreactive type, so that it appears justified to correlate the reactivity of granules with vasopressin, consequently to distinguish neurones and fibres containing vasopressin from those in which oxytocin is quantitatively the main hormonal peptide. This conclusion is strongly supported by the fact that percentages of reactive and negative endings, as determined on this basis in the posthypophysis of normal rats from two different strains, are in good agreement with biochemical data reported in the literature.

Animals↗
PubMed1982

Applications of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections.

The protein A-gold (pAg) complex, a useful reagent for electron microscopic localization of antigens in thin sections, is tested for its suitability as second step reagent in light microscopic immunohistochemistry. In addition, the preparation of colloidal silver, its complex formation with staphylococcal protein A and the application of the protein A-silver complex for antigen localization in paraffin sections is reported. The antigens were visualized in a two-step technique with specific antisera in the first incubation step and pAg or pA-silver as a general second step reagent. The pAg complex gives a red coloration of antigenic sites, whereas the pA-silver stained yellow. The contrasting color provided by the two immunocolloids allowed localization of two antigens in the same section. No color mixing occurred, showing that removal of the antibodies of the first staining sequence is unnecessary. Staining is virtually permanent with the light microscopic immunocolloid method. It is concluded that pAg and pA-silver complexes are useful as general second step reagents for the localization of a variety of antigens in paraffin sections.

Animals↗
PubMed1981

Ultrastructural and cytochemical study on the enzyme gland of the foot of a mollusc.

The enzyme gland of the foot of the mussel Mytilus has been so far considered a gland producing and exporting a phenol oxidase catalysing the general tanning processes of byssus threads. In contrast, the present study shows that this gland produces mainly secretory granules which form the cortical layers of byssus threads. Cytochemical methods at the ultrastructural level (phosphotungstic acid at low pH, silver methenamine, periodic acid-thiosemicarbazide-silver proteinate, silver methenamine for sulphur-rich proteins demonstration) and enzyme digestion tests (pepsin, trypsin, alpha-chymotrypsin) indicate that secretory granules contain glycoproteins rich in sulphydryl groups and in aromatic amino acids. The cytochemical demonstration of phenol oxidase shows that enzyme activity is present in Golgi complex, whereas it is absent in secretory granules. For this reason, phenol oxidase does not seem to be exported and utilized for tanning of byssus threads, but it might rather be involved in the elaboration and tanning of the content of the secretory granules in the enzyme gland itself.

Amino Acids↗
PubMed2002

Recovery of intact proteins from silver stained gels.

Silver stained proteins of a wide molecular weight (MW) range (20-116 kDa) were successfully recovered by both electroblot and electroelution. The recovery was demonstrated for nanogram loads of proteins separated by SDS-PAGE and visualized by silver staining methods compatible and incompatible with mass spectrometry (MS). It was shown that the alcohol/acid and glutaraldehyde fixation steps present in a number of staining procedures did not prevent recovery of intact proteins from gels. It was found that the recovery of intact proteins from silver stained gels was substantially increased upon pre-equilibration in a buffer containing the reducing agent, dithiothreitol (DTT). The effect of destaining on the recovery of silver stained proteins was also investigated. Comparable recovery of intact proteins within a wide MW range from silver stained gels with and without destaining step was demonstrated. Recovery of model proteins from gels visualized using silver staining method compatible with MS showed 52 to 76% yield of that from the unstained gel, depending upon method of the transfer. Comparison of the recovery of intact proteins from gels visualized using other staining procedures was also made. The above findings have implications as to the supposed irreversible nature of protein "fixation" inside polyacrylamide matrix, and confirm lack of binding of proteins in the gel to metal silver deposited on its surface. This method has the potential to be suitable for direct characterization of proteins by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) without additional purification steps.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals↗
PubMed1996

Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Amino Acid Sequence↗
PubMed2006

Time-based analysis of silver-stained proteins in acrylamide gels.

Silver staining of proteins after PAGE often remains the method of choice in many laboratories. Nevertheless, it is known that quantification of protein levels is keenly restricted to a small range of protein concentrations leading to an over- or underestimation of protein amounts. To overcome this, a time-based analysis method was developed to avoid the saturation effect of the silver-staining reaction, thus resulting in an improved dynamic range of the gel image produced and therefore better quantification of proteins. Instead of the well-known end-point image analysis, gray intensities of time series images of a developing gel are determined and times until a threshold gray value is reached are calculated. These times are used to calculate a new grayscale image which can be analyzed using commercial image processing software.

Animals↗
PubMed1992

A comparison between low background silver diammine and silver nitrate protein stains.

Several methods of silver staining of proteins after sodium dodecyl sulfate-electrophoresis in polyacrylamide gels were compared. The most rapid methods were found to be less sensitive than more time-consuming methods. Among the long methods, those using glutaraldehyde treatment of the gel and silver diammine complex as the silvering agent were found to be the most sensitive, at the expense of the use of a modified polyacrylamide matrix and higher silver concentrations.

Ammonia↗
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↗
PubMed1991

Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes.

We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins.

Blotting, Western↗