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A new method for destaining blots stained with colloidal gold is described. After destaining and restaining, artifacts and discolorations caused by the colloidal gold can often be removed. The silver produced by silver enhancement can also be selectively removed without affecting the gold stain.
A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60% sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol, octadecylrhodamine, and the adsorbed gold colloid itself. The isolates routinely contained approximately 60% of these tags, enriched approximately 15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity, and ease.
A colloid titration technique has been used to estimate the surface charge content of three distinct cell types of differing surface charge characteristics, i.e., human red blood cells, the surface of which is studded with sialic acid residues, endothelial cells which are surrounded by a thick glycocalyx, and chondrocytes which, when grown at high cell density for several weeks, synthesize a dense pericellular matrix similar to that observed in cartilaginous tissues. Estimates of the charge content obtained for human erythrocyte ghosts and cultured endothelial cells are in good agreement with the charge determined by chemical analyses. On the other hand, the charge at the surface of chondrocytes represented only a fraction of that calculated from measurements of the glycosaminoglycan content (15% by Day 12 in culture); close correlation between charge and chemical amount could be obtained only after digestion of the cell layer with collagenase and papain indicating the possible electrostatic involvement of the negative groups of the glycosaminoglycan chains with basic proteins. Thus, the colloid titration technique may provide a simple and sensitive tool to study the interactions occurring between the extracellular matrix components while the matrix is being formed and to establish the chemical nature of the molecules contributing to the cell surface charge.
The binding and endocytic uptake of low-density lipoprotein (LDL) particles by cells, transiently or permanently transfected with the human LDL receptor cDNA, was investigated, under different situations, using colloidal gold-LDL conjugates. The amount of gold associated with the various cells, which bind and internalize LDL to different extents, was estimated by inductively coupled plasma-MS. In all cases, the existing differences in LDL binding and uptake were clearly detectable with this procedure. We conclude, therefore, that inductively coupled plasma-MS provides an appropriate assay system for the rapid quantitation of these processes. This procedure also recognizes differences in LDL receptor expression in human lymphocytes and, therefore, it could be of value for the differential diagnosis of LDL receptor defects in familial hypercholesterolemia in various cell types. In addition, this easily performed methodology can also be applied to a variety of other problems requiring quantitation of colloidal gold associated with cells.
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.
Irreversible adsorption of colloid particles and globular proteins at heterogeneous surfaces was studied theoretically. The substrate surface was created by covering a uniform surface by coupling sites (active centers) of a desired coverage. In contrast to previous studies concerned with disks, in our simulations the centers were modeled by spheres having a size smaller than that of the adsorbing particles. Adsorption was assumed to occur due to short-ranged attractive interactions if the colloid particle contacted the center. The Monte-Carlo-type simulations enabled one to determine the initial flux, adsorption kinetics, jamming coverage, and the structure of the particle monolayer as a function of the site coverage and the particle/site size ratio, denoted by lambda. It was revealed that the initial flux increased significantly with the site coverage theta(s) and the lambda parameter. This behavior was quantitatively interpreted in terms of the scaled particle theory. It also was demonstrated that particle adsorption kinetics and the jamming coverage increased significantly, at fixed site coverage, when the lambda parameter increased. Practically, for alpha=lambda(2)theta(s)>1 the jamming coverage at the heterogeneous surfaces attained the value pertinent to continuous surfaces. The results obtained prove unequivocally that the spherically shaped sites are much more effective in binding particles than the disk-shaped sites considered previously.
Additive interactions and their effects on electrokinetic properties of colloidal alumina suspension are presented in this study. Dibasic ammonium citrate (DAC), natural egg albumin, and octanol-2 are chosen as additives. The surface charge behavior of a 5% (w/v) colloidal alumina suspension in the presence of these additives has been studied in detail. The optimum dosage of DAC to have a stable suspension is found to be 3 mg/g of alumina whereas it is 105 and 164 mg/g of alumina for albumin and octanol-2, respectively. Albumin was found to be a better dispersant than DAC and interacts more strongly than DAC with the surface of alumina, while octanol-2 shows physisorbing characteristics. Turbidity measurements indicate that albumin and DAC interact. When the DAC : albumin molar ratio exceeds 10, the turbidity of the solution is higher than the turbidity of pure albumin, indicating the formation of complexes.
The occurrence of fluid-phase endocytosis in Sertoli cells as well as epithelial cells of the rete testis and ductuli efferentes was demonstrated by use of horseradish peroxidase combined with colloidal gold (HRP-G) as a tracer. The characteristic electron dense spherical colloidal gold particles facilitate the localization of the HRP-G complex in pinocytotic vesicles, multivesicular bodies, and lysosomes of these epithelial cells. With this method, which does not require the use of diaminobenzidine, a clear distinction can be made between peroxidases from endogenous and exogenous origins.
Twenty-four patients with cervical cancer stage Ib were examined by pelvic lymphoscintigraphy with 99mTc-colloid prior to operation. Four of the patients had histologically verified lymph node metastases. No difference in the lymphoscintigrams was found between the patients with pelvic metastases and those without. The removed involved lymph nodes did not contain less radioactive colloid than the histologically normal nodes. In two involved lymph nodes the uptake of radioactivity was far above any of the noninvolved nodes.
A technique for the measurement of the rate of uptake of 99mTc labelled sulphur colloid in the liver using a gamma camera and computer is described. The mathematical model showing the factors affecting the rate of uptake, and the results of the measurements in a series of normals and patients with a variety of liver diseases are presented. The range in cirrhosis was found to be significantly lower than normal and the range in acute liver injury significantly higher. The technique can easily be incorporated into a standard colloid liver imaging procedure and is considered to be a valuable adjunct to diagnosis using the static images.
A 44-year-old man developed severe permeability pulmonary edema associated with septic shock after near-drowning. Colloid osmotic pressure (COP) of deep tracheal fluid was persistently higher than plasma COP. Tracheal/plasma COP ratio reached a peak value of 1.75. A massive colloid infusion of 5% protein solution was incriminated in the development of this markedly elevated COP in the tracheal fluid.
A procedure for the radiochemical purity control of colloid and particulate 99mTc-labeled radiopharmaceuticals is described. The proposed technique is based on the use of two chromatograms, using in both, 15% H3PO4 as solvent and impregnated glass fiber media (Gelman ITLC type SG) as stationary phase. A pretreatment of the radiopharmaceutical with 6 N NaOH is involved prior to one chromatographic run. The procedure is fast and the different species (free pertechnetate, 99mTc-Sn-colloid and labeled 99mTc) in a 99mTc-labeled radiopharmaceutical can be determined accurately and with reliability.
Markers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wide acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat beta-lactoglobulin (beta L) tritiated in vivo. When this protein was modified chemically by iodination with 125I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of beta L, a maximum of 13-14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that betas L was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-beta L antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 beta L molecules per particle could be completely bound by immobilized anti-beta L antibodies. Preliminary data indicated that the energetics of adsorption of beta L onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.
The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M chi and L chi values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L chi values were reliable, whereas the M chi values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L chi values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.
Concanavalin-A-colloidal gold (Con-A-G) complex and adenylate cyclase activity were detected simultaneously in electron microscopic preparations of human fibroblast cultures, by a combined histochemical technique. The colloidal gold particles appeared as round bodies which could be readily differentiated from the amorphous product of the adenylate cyclase enzyme reaction. The combined technique makes possible the simultaneous visualization of the bound ligand (i.e. of its binding site), and of the enzyme activated by the ligand. Treatment of the cells with Con-A accounted for a considerable increase in intracellular adenylate cyclase activity. The activity increase was disproportionally greater than the amount of bound ligand, and it also appeared in localizations showing no indication of ligand binding. Treatment of the fibroblasts with Con-A was followed by internalization of the ligand and the enzyme inside at least seemingly segregated vesicles.
Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal 125I-IgE antibovine milk beta-lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1-8.8 (28 nm in particle diameter). At low coverage of the particles (less that or equal to 5 molecules per particle), the isotherm was independent of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2-5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mapping of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4 degrees C. Surface clusters developed when the cells were incubated at 37 degrees C.
A new method is reported for the preparation of colloidal gold particles with diameters ranging between 5 and 12 nm. The initial gold particle population, with an average diameter of 5.6 +/- 0.9 nm, is prepared by reduction of chloroauric acid with white phosphorous. An increase in particle diameter by growth is obtained by reduction of chloroauric acid with white phosphorous in the presence of colloidal gold particles. The labelling efficiency of these gold particles, conjugated with protein A, in indirect immunolabelling experiments is investigated by labelling of beta-galactosidase on ultrathin cryosections of Escherichia coli cells. We demonstrate that the labelling efficiency is at least dependent on particle diameter, probe concentration and preparation method. In addition it is shown, that with this new method, gold particle populations can be prepared with minor overlap in diameter spreading. Therefore these gold probes are suitable for qualitative double labelling experiments. The quantitative aspect of immunolabelling is discussed.
The thick sialoglycoprotein coat of the glomerular epithelial cells is a part of the glomerular filtration barrier. It can be demonstrated in paraffin histoslogy by means of the colloidal iron procedure. We have studied the special requirements of tissue processing to obtain reproducible results by this staining method. The requirements include proper fixation, avoiding heat during sectioning and ample rehydration. We have further tested a colloidal iron-PAS procedure for simultaneous demonstration of the glomerular epithelial coat and the basement membrane.