A comparison of silver staining methods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing

The application of silver staining methods to the detection of proteins on ultrathin isoelectric focusing gel systems requires the optimization of many steps in the procedure in order to obtain reproducible staining of proteins with acceptable levels of background. Three different methods which have been reported for detecting proteins by silver staining in sodium dodecyl sulfate-polyacrylamide gel systems were investigated. A major problem with staining ultrathin isoelectric focusing gels was found to be surface staining that was associated with gels cast on support films. A modification of the method of Poehling and Neuhoff (H.-M. Poehling and V. Neuhoff, 1981, Electrophoresis 2, 141-147) was found to give the best results.     

A new silver staining apparatus and procedure for matrix-assisted laser desorption/ionization-time of flight analysis of proteins after two-dimensional electrophoresis

We report on a new silver stain especially developed for staining large gels (25 cm x 20 cm) from the Hoefer ISO-DALT system for matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of proteins. The staining protocol can be summarized as follows: the gels are sensitised in tetrathionate/potassium acetate solution and washed several times in distilled water. After impregnation with silver nitrate, the silver is reduced in the presence of potassium carbonate, thiosulphate and formaldehyde. The staining procedure is stopped with Tris/acetate after which the gels are rinsed and stored in water before spot picking for MALDI-TOF analysis is performed. This protocol has several advantages over existing ones. The gels are stained in a new apparatus that reduces gel handling to a minimum thus also reducing the contamination with keratins to a minimum. The development times in potassium carbonate are very long (up to 40 min) thus improving batch-to-batch reproducibility. Only the surface of the proteins is stained and the silver can be oxidized, thereafter MALDI-TOF can be performed with protein loads as little as 100 micrograms per gel.     

Autometallography

Summary The autometallographic procedure represents a new technique that can substitute for the normal methods of physical development (PD). The physical developer (a solution of reducing substance, silver salt and protection colloid) is replaced by a photographic emulsion and chemical developer. Accumulations of gold, silver, metal sulphides and metal selenides can be amplified by the present technique.Tissue sections placed on glass slides are covered by a silver bromide containing emulsion, dried and exposed to a chemical developer. After development the emulsion is either removed or cleared and the sections are counterstained and embedded.The autometallographic procedure can also be applied to ultrathin sections.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

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.     

A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver

A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments.     

Quantitation of protein and DNA in silver-stained agarose gels

A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to 100-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment sizes studied.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Summary Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Costaining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Co-staining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

Protein analysis on two-dimensional polyacrylamide gels in the femtogram range: use of a new sulfur-labeling reagent

An ultrasensitive staining procedure for two-dimensional polyacrylamide gels of protein homogenates has been developed. It combines the use of ultrathin gels and the labeling of proteins by a 35S-labeling reagent.     

Ultramicrodetection of proteins in polyacrylamide gels.

Here we report the development of a highly sensitive procedure to detect proteins within separation matrices which should facilitate the characterization of rare proteins. The procedure is based on photochemical reactions where very low amounts of silver are deposited around proteins and in a series of steps are converted to silver sulfide. When this conversion is carried out in the presence of [35S]thiourea the resulting radioactive silver sulfide allows detection down to femtogram quantities of protein. In this work we applied the above principle to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, thus not influencing physical and chemical parameters which are important for separation. This procedure should find application in any technique where detection of very low or limited amounts of proteins are required.     

Simple, sensitive, and quick protocol to detect less than 1 ng of bacterial lipopolysaccharide

Detection and quantitation of lipopolysaccharide (LPS) are important for quality assurance of pharmaceutical, biopharmaceutical products and for several research and industrial aspects. The widely available assays for LPS detection are the normal, or chromogenic, and the synthetic LAL (Limulus amebocyte lysate). Unfortunately, both assays are expensive and could not distinguish between different types of bacterial LPS, while LPS silver nitrate staining requires more than 20 hr and can only detect 5 ng LPS. The current modified protocol was able to detect less than 0.5 ng LPS in a polyacrylamide-urea gel. The procedure is rapid, inexpensive, and reproducible. It depends on introducing two modifications to improve the staining sensitivity in sample buffer and staining procedure. The results revealed that excluding the reducing agent sucrose to use instead glycerol, and replacing SDS with DOC, as well as incorporation of 4 M urea in stacking and separating gels, increased the sensitivity up to 150 pg. In summary, the gels were fixed, carbohydrates moieties were oxidized to create active aldehydes, and the gels were silver stained using non-ammoniacal silver nitrate.     

A Simple and Highly Reproducible Staining Method for Fungi and Other Polysaccharide-Rich Microorganisms in Animal Tissues

A newly devised, simple and highly reproducible method for fungal staining is reported. Grocott's method, in which methenamine-silver nitrate solution is employed, has been widely used for the staining of fungi in tissue sections, but it frequently produces heavy background staining because of sudden and progressive reaction in the methenamine-silver nitrate solution. We therefore replace the latter solution with an ammoniacal silver nitrate solution. This new method yields more consistent results in fungal staining without background staining, since the reaction time in die ammoniacal silver nitrate solution is prolonged. The present method is considered superior to Grocott's method with regard to its simplicity and reproducibility.     

A simple and highly reproducible staining method for fungi and other polysaccharide-rich microorganisms in animal tissues

A newly devised, simple and highly reproducible method for fungal staining is reported. Grocott's method, in which methenamine-silver nitrate solution is employed, has been widely used for the staining of fungi in tissue sections, but it frequently produces heavy background staining because of sudden and progressive reaction in the methenamine-silver nitrate solution. We therefore replace the latter solution with an ammoniacal silver nitrate solution. This new method yields more consistent results in fungal staining without background staining, since the reaction time in the ammoniacal silver nitrate solution is prolonged. The present method is considered superior to Grocott's method with regard to its simplicity and reproducibility.     

Cerium as amplifying agent--an improved cerium-perhydroxide-DAB-nickel (Ce/Ce-H2O2-DAB-Ni) method for the visualization of cerium phosphate in resin sections

A new visualization (Ce/Ce-H2O2-DAB-Ni) procedure for cerium (Ce III) phosphate in semithin and ultrathin plastic sections (Epon 812, Lowicryl K4M, glycol methacrylate) of rat kidney tissues that had been incubated before embedding for the demonstration of phosphatases (alkaline and acid phosphatase, 5(1)-nucleotidase, Mg-dependent ATPase) is described. For this purpose the hydrophobic Epon resin was removed in NaOH-ethanol solution, whereas the hydrophilic Lowicryl and methacrylate sections did not required any etching. The primary reaction product Ce III-phosphate was amplified in a Ce III-citrate solution, subsequently oxidized with H2O2 and then visualized in a H2O2 containing DAB-nickel medium (Ce IV-perhydroxy induced DAB polymerization principle). The method yielded a very clear localization of enzyme activity. The final reaction product (DAB-nickel polymers) in 0.5 - 2.0 microns semithin sections is blue-black; the background staining is completely prevented. An increase of the staining contrast was obtained by posttreatment with OsO4 (osmium black formation). Furthermore, the enzyme reaction product could be demonstrated in 40 nm thick ultrathin sections by silver intensification, which utilized the high argyrophilia of the polymerized DAB-nickel complexes. This procedure replaces the earlier published technique.     

Enhancement of the periodic acid--Schiff (PAS) and periodic acid--thiocarbohydrazide--silver proteinate (PA-TCH-SP) reaction in LR white sections

LR White is a well-suited resin for the demonstration of carbohydrates with the PAS or PA-TCH-SP reaction in semithin and ultrathin sections. The intensity of these reactions can be greatly enhanced by using 3 steps in tissue preparation, either singly or in combination: 1) The PAS reaction in semithin sections turns out stronger after partial (70% ethanol) than complete (100% ethanol) dehydration of the tissue before its transfer to 100% LR White. 2) Silver enhancement of the PA-TCH-SP reaction product can simply be effected by physical development of ultrathin sections (PA-TCH-SP-SE reaction). Least precipitates are formed in this procedure, when sections are mounted on uncoated gold grids, processed for cytochemistry, and thinly coated with carbon in the end. 3) The use of hot silver proteinate (50 degrees C) plus strong silver enhancement (15-20 min silver lactate developer) reveals minute concentrations of TCH-labelled aldehyde groups in the tissue that do not react with silver proteinate at room temperature.--Silver enhancement and the use of hot silver proteinate do not depend on LR White, but may also be applied to ultrathin sections of tissue embedded in other resins.     

Colloidal Silver Staining of Electroblotted Proteins for High Sensitivity Peptide Mapping by Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry.     

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.     

Robust estimation of standard curves for protein molecular weight and linear-duplex DNA base-pair number after gel electrophoresis

An accurate procedure for estimating linear-duplex DNA base-pair numbers and protein molecular weights after electrophoresis in single concentration gels is presented. A robust modified hyperbola was found to be superior for determining molecular weights and base-pair numbers for a set of known standards when compared with the conventional log transformation and a similar hyperbolic model. We describe the use of a soft laser-scanning densitometer to measure band-migration distances of wet, stained polyacrylamide gels for proteins and photographic negatives of agarose gels containing DNA stained with ethidium bromide. This automated densitometric method was more accurate than existing methods. A BASIC computer program detailing the procedure is included.     

Selective silver staining of urease activity in polyacrylamide gels

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.