Ss blood Group Associated PAS-staining polymorphism of glycoprotein 3 from human erythrocyte membranes

Summary It is shown by discontinuous sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis of human red cell membranes, followed by periodic acid Schiff (PAS) staining and densitometry, that the band PAS-3 (monomeric Ss glycoprotein) exhibits a polymorphism with respect to its staining intensity. In membranes of the genotype SS the staining intensity of this band is about 1.5 times higher than in ss membranes. The experimental error of the method does not permit one to decide, whether membranes of blood type Ss exhibit an intermediate staining intensity or not. The SDS electrophoretic molecular weight of PAS-3 is the same for SS, Ss, or ss membranes. The ratio of PAS to coomassie blue staining intensities, which are a measure of sialic acid and protein content, respectively, are equal for PAS-3 in glycoprotein preparations from SS and ss erythrocytes. These data indicate that the above phenomenon reflects a difference in the glycoprotein content between SS and ss membranes.     

FerriDye: colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

The Lipids Stained by the Periodic Acid-Schiff Technic

Unsaturated periodic acid-Schiff (PAS) stainable lipids of renal basement membranes are soluble in lipid solvents and do not add to the PAS staining in paraffin embedded sections. These lipids contribute to the staining of basement membranes in frozen sections. Pure sphingomyelin is stained by the PAS method if the oxidising solution is sufficiently acid and the time allowed for periodic oxidation is sufficient. This staining is considered to depend on splitting of the amide link between sphingosine and the fatty acid which leaves the 1-amino-2-hydroxyl grouping of sphingosine available for reacting with the periodic acid.     

India ink staining of proteins on nylon and hydrophobic membranes

India ink was found to be an acceptable stain for proteins blotted or dotted onto positively charged nylon or hydrophobic membranes. The hydrophobic membrane, Immobilon, was an outstanding matrix for binding proteins and displayed low levels of background staining. The least amount of protein detected by india ink staining was between 1.0 and 10 ng. India ink staining of proteins on nylon membranes is an easy, inexpensive, and quick method for the unequivocal detection of both standards and unknowns in the same blot. However, inks, ink concentrations, fixing conditions, staining times, pH, washing conditions, and membrane lots all need to be controlled to achieve maximum sensitivity for protein detection following india ink staining.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Use of tissue-free glycol methacrylate sections as semi-permeable membranes: a simple way to shorten incubation times and to improve localization in enzyme histochemistry

Placing 2-microns sections of tissue-free glycol methacrylate on top of tissue sections is a simple way of forming semipermeable membranes to enhance enzyme histochemical staining. For demonstrating alkaline phosphatase in glycol methacrylate-embedded kidney by a standard azo dye method, such membranes enabled incubation times to be reduced to 1-2 hr, with azo dye reaction product being more crisply localized as compared to sections stained without membranes. Such effects are possible because the membranes are highly permeable to small molecules (e.g., substrate and diazonium salt), slightly permeable to molecules of moderate size (e.g., the final reaction product), and impermeable to large molecules (e.g., alkaline phosphatase and other tissue biopolymers). The implications of these findings for enzyme histochemistry and for enzyme-labeled antibody staining are discussed.     

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Contrasting of Lowicryl K4M thin sections.

A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.     

Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes

The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes.     

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.     

A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin-biotin-peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.     

Fast and efficient method for detection and estimation of proteins

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.     

A new method for the isolation of renal basement membranes.

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent N-lauroyl sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues. The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

Post-electrophoretic identification of oxidized proteins

The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold.     

Ultrastructural localization of glycoproteins in rabbit fibroblasts altered by Herpes simplex virus type 1 infection

The periodic acid-thiocarbohydrazide (SO2)--OsO4 method was used to examine the distribution of glycoproteins in rabbit fibroblast cells infected with Herpes simplex virus type 1. In non-infected cells, a low level of staining was seen over the plasma membrane and the membranes of the Golgi apparatus. At 17 hr post-infection, the intensity of reaction was increased to include not only a relatively heavy staining of the plasma membrane, including the numerous microvilli characteristic of infected cells, and of the newly proliferated Golgi membranes, but also the envelopes of intracytoplasmic and extracellular virions. A very faint but only occasional staining also was associated with the virus-induced reduplications of the inner nuclear membrane and the envelopes of associated enveloping nucleocapsids. We suggest that such differences in the intensity of staining may be related either to the amount of glycoproteins or to the sequential maturation of the viral glycoproteins. We also observed that the structurally modified portions of the Golgi membranes at the position where intracytoplasmic naked nucleocapsids bud into the Golgi cisternae usually exhibit a more intense reaction for glycoproteins than do the adjacent portions of the Golgi membranes. This supports the evidence for an envelopment of nucleocapsids in the cytoplasm, but it does not indicate whether this event obligatorily follows or only occasionally takes the place of the envelopment of nucleocapsids at the inner nuclear membrane. In either event, the envelopes of all mature virions exhibit a prominent reaction to glycoproteins.     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.     

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.     

Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry

A new triple fluorescent staining method was developed to evaluate frozen-thawed dog spermatozoa. This method was used to compare functional parameters of canine spermatozoa cryopreserved using 2 different freezing-thawing protocols. One ejaculate from each of 10 dogs was split into 2 aliquots and processed using the Andersen method or the CLONE method. Semen samples were evaluated immediately after thawing and after 3 h of incubation at 37 degrees C. Plasma membrane integrity and acrosomal status of the spermatozoa were evaluated simultaneously by flow cytometry using a combination of 3 fluorescent dyes: Carboxy-SNARF-1 (SNARF), to identify the live spermatozoa; propidium iodide (PI), which only stains dead cells or cells with damaged membranes; and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA), which binds to the acrosomal content of spermatozoa with damaged plasma and outer acrosomal membranes. The accuracy of this new staining method in quantifying the proportions of live and dead spermatozoa by flow cytometry was evaluated by comparing it with the staining technique using carboxyfluorescein diacetate and propidium iodide (CFDA-PI), which yielded high correlation coefficients. The triple-stained sperm samples were also analyzed by epifluorescence microscopy, and both methods proved to be highly correlated. Post-thaw progressive motility and plasma membrane integrity were similar for the 2 freezing procedures, but the proportion of damaged acrosomes after thawing was lower using the Andersen method and the spermatozoa had a higher thermoresistance. This new triple staining method for assessing canine sperm viability and acrosomal integrity provides an efficient procedure for evaluating frozen-thawed dog semen samples either by flow cytometry or fluorescence microscopy.