Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

A rapid and easy method for staining lipopolysaccharides with ethidium bromide is described. Lipopolysaccharides could be visualized by ethidium bromide with almost the same sensitivity as found with the silver-staining method in less than 30 min. The ethidium bromide-staining method was particularly suitable for staining lipopolysaccharides possessing acidic O-specific polysaccharides, which were poorly visualized by silver staining.     

Tryptic peptide analysis of nanogram quantities of proteins: radioiodination of proteins detected by silver staining in polyacrylamide gels

The silver-staining procedure for detecting proteins in polyacrylamide gels has been modified so that the polypeptides remain suitable for subsequent radioiodination and tryptic peptide analysis. The procedure, which involves a silver-staining/destaining protocol that minimizes crosslinking, is more rapid than many other methods, and can detect as little as 1 ng of protein. Following elimination of silver, the proteins can be radioiodinated and digested with trypsin by a modification of the method described by J. H. Elder, R. A. Pickett, J. Hampton, and R. A. Lerner (1977, J. Biol. Chem. 252, 6510-6515). Together, these improvements have increased the sensitivity of the tryptic peptide mapping technique by three orders of magnitude and thereby enabled us to perform reproducible structural analysis of femtomolar quantities of proteins.     

Detecting proteins containing 3,4-dihydroxyphenylalanine by silver staining of polyacrylamide gels.

Proteins in which some or all of the tyrosine side chains are post-translationally modified to dihydroxyphenylalanine have been found in several invertebrate phyla. In this paper we describe the unusual silver-staining properties of these 3,4-dihydroxyphenylalanine (Dopa)-proteins in silver-stained polyacrylamide gels. Our evidence suggests that the rapid silver staining of these proteins is due to the 3,4-dihydroxyphenol ring which is a highly effective reducing agent in the alkaline development conditions used in the final step of most silver-staining procedures. Normal proteins comprising the standard 20 amino acids and tyrosine on its own, do not reduce silver under these conditions. Pretreatment of the gels with acid-dichromate solutions abrogates the rapid staining of the Dopa-proteins. This rapid silver-staining technique will facilitate the rapid screening of many additional organisms for Dopa-proteins using sodium dodecyl sulfate gels and small amounts of tissue.     

Ultrasensitive silver-stain method for the detection of proteins in polyacrylamide gels and immunoprecipitates on agarose gels

The highly sensitive silver-stain procedure for the detection of proteins in polyacrylamide gels has been revised and simplified using a single-step silver ion reduction after suitable treatment of proteins with bifunctional aldehyde. Washing steps were eliminated and excellent reproducibility of results was achieved. Sensitivity obtained using this procedure was at least equal to that obtained with the original one. Use of the present silver-staining methods has been extended to the quantitative analysis of immunoprecipitates on agarose gels, with a good increase of sensitivity and excellent increase of resolution when compared to the Coomassie blue stain.     

Distribution of silver-stained interphase nucleolar organizer regions as a parameter to distinguish neoplastic from nonneoplastic reactive cells in human effusions

The distribution of interphasic nucleolar organizer regions (NORs) was studied in cytologic preparations of human serous effusions in order to differentiate malignant cells from nonmalignant reactive cells. The study was carried out on 80 cases of metastatic adenocarcinoma, 10 cases of mesothelioma, 10 reactive pleural effusions and 5 peritoneal washings. Visualization of NORs at the light microscopic level was obtained using a silver-staining technique for acidic proteins selectively associated with NORs. The morphologic data were also statistically evaluated by means of an automated image analyzer. The quantity of silver-stained NORs was higher in cancer cells (both mesothelioma and adenocarcinoma) than in reactive mesothelial cells. Moreover, NORs were more irregularly distributed within the nucleoli and were more variably sized in cancer cells than in reactive mesothelial cells.     

Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells.

Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.     

Generation of recombinant antibodies against orchardgrass Acidic nsLTP-like proteins

Embryogenic and non-embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.) secreted into the culture medium a set of proteins, among which low molecular mass (11/12 kDa) proteins were found. However, only the 11/12 kDa proteins from the embryogenic suspension cultures reacted specifically with an antiserum raised against the carrot EP2 non-specific lipid transfer protein (nsLTP). Two-dimensional (2-D) electrophoretic analysis revealed that the extracellular nsLTP-like proteins from the embryogenic lines were acidic proteins, with pI values ranging between 4.3 and 6.4, and the 11/12 kDa proteins of the non-embryogenic lines were basic ones (pI 8-9.3). This is only the second case to report on the accumulation of extracellular acidic nsLTP-like proteins in the culture medium during somatic embryogenesis. A naive phage display Griffin1. library was used to select single-chain phage antibodies, which specifically bind to acidic nsLTP-like proteins. Nine phage clones were selected after four rounds of biopanning of the target proteins blotted on a nitrocellulose membrane. Three soluble monoclonal single-chain phage antibodies, expressed in the non-suppressor E. coli strain HB2151, were purified by metal affinity chromatography and found to be highly specific for the acidic nsLTP-like proteins from the embryogenic suspension cultures. The application of the selected monoclonal antibodies for localization and elucidation of the role of the acidic nsLTP-like proteins in vivo is discussed.     

嗜肺军团菌简易鞭毛染色法及在不同培养基上鞭毛的生长观察

本文报道了一种较为简易、快速的嗜肺军团菌 Legionella pneumophila 鞭毛染色法,并探讨了嗜肺军团菌在两种培养基(BCYE、NYE)上鞭毛的生长情况。实验结果提示这种鞭毛染色法有助于军团病的病原学诊断。     

Behaviour of nucleolus during mitosis

The aim of the present work was to study the distribution and the behaviour of the silver-staining nucleolar organizer region (Ag-NOR) proteins at the ultrastructural level during interphase and mitosis in five human and murine cancerous cell lines each characterized by a typical nucleolar morphology. During interphase the Ag-NOR proteins are restricted to the fibrillar centres (F.C.) and/or to the dense fibrillar component (D.F.C.). During prophase the silver-staining components come into close contact with some chromosomes and are arranged with a typical polarity: chromosome, F.C. and D.F.C. Then F.C. and D.F.C. together form roundish silver-stained structures and integrate in part within indentations at the periphery of the metaphase chromosomes. During anaphase and telophase large and small spherical silver-staining structures may be seen. They correspond respectively to the metaphase NORs and to numerous structures which appear de novo within ribonucleoprotein (RNP) material localized between the chromosomes. During late telophase the number of the small silver-staining structures decreases whereas the size of the larger ones increases. Then the interphase nucleoli recover their typical shape. These results suggest that when rRNA synthesis is impaired during mitosis the inactive NORs assume a structure and a localization which are not typical of the cell line. In contrast the F.C. and D.F.C. are probably two aspects of the NORs whose typical distribution, relative to the other nucleolar components, gives the interphasic nucleolus its characteristic morphology.     

Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.

This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction for the amount of antibody remaining on the membrane after the milk block, it was found that acid-bound antibodies were unchanged in biological activity when compared with the same antibodies bound at neutral pH. These results suggest that acid binding of proteins could increase the sensitivity of nitrocellulose membrane assays using a milk block.     

A silver-staining technique for detecting minute quantities of proteins on nitrocellulose paper: retention of antigenicity of stained proteins

A method using a silver-staining procedure to detect minute quantities of proteins on nitrocellulose paper is described. The technique is sensitive enough to detect nanogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose paper by the electrotransfer technique. After the staining procedures, the proteins are shown to retain their antigenic properties.     

Purification and characterization of TNP-specific immunoregulatory molecules produced by T cells sensitized by picrylchloride (PC1F)

T cell-derived TNP-specific factors associated with immunoregulatory activity were obtained by culture of T cells obtained from mice sensitized by skin-painting with picrylchloride. Culture medium was absorbed to TNP-Sepharose and TNP binding proteins were prepared by elution with TNP. The hapten affinity-purified proteins were characterized by size and charge and were found to be acidic 70,000 m.w. polypeptides that occur as monomers or oligomers. Oligomeric proteins interact with factors produced by mice injected with trinitrobenzenesulfonic acid to form factors that suppress specifically the ability of TNP-sensitized T cells to transfer contact sensitivity to TNP. Monomeric (no more than 70,000 m.w.) molecules do not form suppressor factors but can transfer contact sensitivity to TNP. Moreover, reduction and alkylation of oligomeric molecules inactivates their suppressor activity but causes them to be able to transfer contact sensitivity. The results suggest that T cell-derived antigen-specific molecules may have different effector functions dependent on their oligomeric state.     

A comparison of the ribosomal proteins of Drosophila ovary, adult, and embryo

Summary The proteins in the 80S ribosomes of Drosophila melanogaster ovaries and adults have been characterized by two-dimensional polyacrylamide gel electrophoresis. When ribosomal proteins of ovaries and adults were compared with those from embryos, all 3 tissues showed a similar number of proteins. In addition, qualitatively, the electrophoretograms of proteins extracted from the ribosomes of these 3 tissues were found to be indistinguishable. However, apparent quantitative differences in certain acidic proteins were observed between tissues. Using ribosomes from embryos as a standard for comparison, ribosomes from adult flies that were more than 14 days old appeared to have relatively larger amounts of acidic protiens S7 and S9, and relatively smaller amounts of acidic proteins S14 and S25/S27. The transition period occured during the ninth to thirteenth day of adult fly development. Significant differences were not detected between ovarian and embryonic acidic ribosomal proteins. In contrast to the differential ratio of acidic proteins in ovaries, adults, and embryos, a similar distribution of basic proteins was found in these tissues.     

Characterization of interaction sites in the Saccharomyces cerevisiae ribosomal stalk components

The interactions among the yeast stalk components (P0, P1alpha, P1beta, P2alpha and P2beta) and with EF-2 have been explored using immunoprecipitation, affinity chromatography and the two-hybrid system. No stable association was detected between acidic proteins of the same type. In contrast, P1alpha and P1beta were found to interact with P2beta and P2alpha respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-amino-acid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1alpha with P2beta and of P1beta with P2alpha rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.     

Isolation of nuclear acidic proteins from rat tissues. Characterization of acetylated liver nuclear acidic proteins

Nuclear acidic proteins isolated from rat brain, heart, kidney and liver showed similar, complex patterns on electrophoresis in sodium dodecyl sulphate-polyacrylamide gels. The contamination of nuclear acidic proteins by nuclear-membrane acidic proteins was found to the extent of 11%. Incorporation of [(3)H]acetate into the various nuclear acidic proteins in vivo, which were fractionated by polyacrylamide-gel electrophoresis, differed from tissue to tissue. Hydrolysis of these acetylated nuclear acidic proteins with 6m-HCl at 110 degrees C released 70% of the radioactivity, which indicated that labile acetyl groups had been incorporated into these proteins. Analysis of [(3)H]acetate-labelled nuclear acidic proteins revealed two acetylated amino acid residues, N(2)-acetylserine and N(2)-acetyl-lysine. The significance of the role played by nuclear acidic proteins in relation to gene regulation is discussed.     

Structural characterization of the glycinin precursors

Poly(A)-RNAs enriched for glycinin coding sequences were injected into frog oocytes and translated in the presence of either [3H]leucine or [3H]isoleucine. Sodium dodecyl sulfate electrophoresis indicated that radioactive proteins similar in size to the authentic acidic and basic polypeptide components of glycinin were not present among the glycinin-related proteins synthesized. Instead, high molecular weight precursors (Mr = 58,000-67,000) were immunoprecipitated. Unlike disulfide-linked native glycinin complexes which were cleaved by disulfide reduction, products purified from either rabbit reticulocyte lysate or oocyte translation systems were insensitive to reducing agents. The glycinin-related proteins synthesized in the oocyte were 1000 to 2000 daltons smaller than those synthesized in the reticulocyte lysate system. This result, which suggested that the oocyte system had removed NH2-terminal leader sequences of the preglycinin polypeptides, was confirmed by NH2-terminal sequence analysis of proteins synthesized in oocytes. Radioactive label was found exactly at the positions predicted by the NH2-terminal sequences of the acidic polypeptide component of native glycinin. Glycinin precursors, therefore, have an NH2-terminal leader sequence followed by the acidic peptide component and then the basic polypeptide component, joined in peptide linkage.     

Cytospectrophotometric analysis of various color reactions for acid and basis proteins in nerve tissue cells]

Polyvinyl formal films soaked with various concentrations of proteins as well as Carnoy-fixed paraffin sections of rat brain were stained with various colour reactions for acidic and basic proteins. Sufficient specificity, reproductivity, sensitivity and applicability for cytospectrophotometric determinations have been shown for the staining of acidic proteins with Toluidine blue 0 (pH 9.0) or with Fast green FCF (pH 2.6), and for the staining of basic proteins with Alcian blue (pH 10.0), or with Fast green FCF (pH 8.2). Importance of cytophotometric analysis of individual protein fractions is outlined for the functional cytochemistry of the nervous system.     

Studies on the relationship between the degradative rates of proteins in vivo and their isoelectric points

Acidic proteins tend to be degraded more rapidly than neutral or basic proteins in rat liver, skeletal muscle, kidney and brain and in mouse liver and skeletal muscle. We now report a similar relationship among soluble proteins from rat lung, heart and testes, and from human fibroblasts and mouse-embryo cells grown in culture. These findings indicate that the correlation between protein net charge and degradative rate is a general characteristic of intracellular protein degradation in mammals. This relationship between isoelectric point and half-life appears to be distinct from the previously reported correlation between subunit molecular weight and protein half-lives. The more rapid degradation of acidic proteins does not result from their being of larger molecular weight than neutral or basic proteins. Furthermore, proteins within specific isoelectric point ranges still exhibit a relationship between subunit size and half-life. Finally, a group of membrane or organelle-associated proteins that are insoluble in phosphate-buffered saline and water but soluble in 1% Triton X-100 exhibit a correlation between size and half-life, but not between net charge and half-life. The biochemical reasons for the relationship between protein isoelectric point and half-life are unclear, although several possible explanations are presented. It is not due to a greater sensitivity of acidic proteins to proteolytic attack since experiments with a variety of endoproteinases, including trypsin, chymotrypsin, Pronase, papain, chymopapain, Staphylococcus aureus V8 proteinase, pepsin and lysosomal cathepsins from rat liver, have failed to demonstrate more rapid digestion of acidic proteins.     

NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.