Partial purification and characterization of Schistosoma mansoni soluble egg antigen with Con A-Sepharose chromatography

A crude preparation of Schistosoma mansoni soluble egg antigen (SEA) was subjected to affinity chromatography with concanavalin A (Con A) bound to Sepharose 4B. The resulting Con A fractions (bound and unbound) were characterized with sodium dodecyl sulfate (SDS) gel electrophoresis, immunoelectrophoresis, immunodiffusion, and lymphocyte blastogenesis techniques. In the fraction that did not bind to Con A there were at least two distinct antigens, and there were also at least two distinct antigens in the fractions that did bind to Con A. With SDS polyacrylamide gel electrophoresis, at least 20 distinct protein bands (Coomassie blue staining) and three glycoprotein bands (PAS reactive) were present in the unbound fractions from Con A chromatography. The bound fractions separated into at least six distinct glycoproteins with SDS electrophoresis. Although both the bound and unbound fractions contained precipitating antigens, only the bound fractions were capable of eliciting lymphocyte blastogenic responses.     

Immunoaffinity purification of Schistosoma mansoni soluble egg antigens

Schistosoma mansoni egg antigens were purified from a heterogeneous mixture of soluble egg antigens (crude SEA) with an immunoaffinity column that consisted of the specific anti-SEA antibodies contained in 16-week S. mansoni-infected mouse serum bound to Sepharose 4B. On sodium dodecyl sulfate (SDS) gel electrophoresis, the purified antigen fraction yielded at least eight bands staining with Coomassie blue and at least five bands staining with Coomaisse blue and at least five bands reacting with periodic acid-Schiff (PAS). All of the proteins in the antigenic fraction appear to contain carbohydrate residues. Upon immunoelectrophoresis the antigen yielded four precipitin arcs. The antigenic fraction isolated by means of the immunoaffinity column was then compared to various fractions obtained from concanavalin A (Con A) chromatography of SEA. The results of Ouchterlony immunodiffusion and immunoelectrophoresis indicate that the antigenic fraction isolated by immunoaffinity purification of SEA contains the major antigens found in the fractions obtained from Con A chromatography of SEA. The results of SDS gel electrophoresis indicate that the major PAS-reacting bands of the antigenic fraction isolated by immunoaffinity purification are found in the 3rd peak (bound fraction) resulting from Con A chromatography of SEA, whereas the major Coomaisse blue-staining band in the isolated antigenic fraction is found in the 2nd peak (unbound fraction) from Con A chromatography of SEA.     

Staining protein in isoelectric focusing gels with fast green

A rapid, simple technique for staining proteins in isoelectric focusing polyacrylamide gels was demonstrated using fast green in 10% acetic acid. Fast green has the distinct advantage of not binding to ampholytes, thus staining only protein. Maximum staining was achieved within 5 min, and bands were visible after 3 to 6 h of destaining. Background stain removal, however, was not complete until 72 h after placing gels in a diffusion destainer. Gel quantitation was demonstrated with actin using fast green and Coomassie brilliant blue R-250. A standard curve prepared with fast green was linear from 0.5 to 8 μg of actin in contrast to Coomassie brilliant blue R-250 which provided linearity from 0.1 to 2.5 μg actin. Application of fast green staining to quantitation of α-actin from cultured muscle satellite cells has been demonstrated.     

Staining properties of bovine low molecular weight hydrophobic surfactant proteins after polyacrylamide gel electrophoresis.

Reports describing polyacrylamide gel electrophoresis patterns of bovine hydrophobic surfactant proteins are not consistent. In this study, we found unusual staining characteristics of these proteins that may explain some of these inconsistencies. Low molecular weight surfactant proteins extracted from bronchoalveolar lavage with organic solvent are partially delipidated with Sephadex LH-20 chromatography using chloroform and methanol. Fractions from the first protein peak are dried under nitrogen then subjected to SDS electrophoresis on 20% polyacrylamide gels. Under nonreducing conditions, silver staining identifies 5- and 26-kDa bands, and Coomassie blue identifies 6-, 12-, and 26-kDa bands. When gels are stained with Coomassie blue then silver, the 5- and 26-kDa bands stain with silver and 6- and 12-kDa bands remain stained with Coomassie blue. If gels are first stained with silver then Coomassie blue, similar results occur. We modified the silver staining protocol by treating gels with dithiothreitol or 2-mercaptoethanol after electrophoresis. With this modification, 5-, 6-, 12-, 26-, and also 17-kDa bands are identifiable. Using the modified protocol and restaining gels previously stained with silver, 6-, 12-, and 17-kDa bands that were not identified previously all became visible. In further experiments, protein bands of 6-, 12-, and 26-kDa that were identified by Coomassie blue were electroeluted under nonreducing conditions. After electrophoresis of the eluted 26-kDa protein, bands of 17-, and 26-kDa under nonreducing, and 8-kDa only under reducing conditions, were apparent by using the modified silver protocol.(ABSTRACT TRUNCATED AT 250 WORDS)     

A sensitive silver stain for proteins in agarose gels

A silver stain for proteins in agarose gels which is at least 10 times as sensitive as Coomassie blue is described. The method is simple to use and is particularly useful for the study of protein bands in the gamma region on electrophoresis of fluids such as cerebrospinal fluid in which the protein concentration is low. It readily detects bands of IgG containing 20 to 40 ng/band (approx 3 to 6 ng of IgG/mm2 of gel).     

一种转移非特异性蛋白带的染色方法

考马斯亮蓝是常用的聚丙烯酰胺凝胶蛋白电泳的染料,利用硝酸纤维素膜(NCF)对染料的吸附作用,将低浓度的考马斯亮蓝(0.025%)染色液直接对NCF上的转移蛋白带进行染色,经实验反复验证.它是一种较好的NCF上转移非特异性蛋白带的染色方法.     

Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immune responses during human schistosomiasis mansoni. VI. In vitro nonspecific suppression of phytohemagglutinin responsiveness induced by exposure to certain schistosomal preparations.

Simultaneous in vitro exposure of human peripheral blood mononuclear cells to phytohemagglutinin-P (PHA) and either soluble schistosomal egg antigenic preparation (SEA) or soluble cercarial antigenic preparation (CAP) obtained from Schistosoma mansoni resulted in decreased responsiveness as compared to exposure to PHA alone. The addition of a soluble adult worm antigenic preparation (SWAP) did not predictably alter PHA responses in this system. The suppression due to in vitro exposure to either SEA or CAP was expressed whether the lymphocyte donors were S. mansoni patients (early infection, chronic, or treated), or uninfected subjects. The degree of suppression was related to the concentration of SEA used, and the timing of exposure. Preexposure to SEA for 3 days before the addition of PHA resulted in more potent suppression. However, a delay in the time of the addition of SEA of 6 and 24 hr after PHA exposure decreased and eliminated, respectively, its suppressive capacity. SEA and CAP were not directly toxic to responding cells, and appeared to exert their nonspecific suppressive influences through T lymphocyte-related mechanisms. It was observed that although these suppressive events could be induced and observed in vitro, the responsiveness of S. mansoni patient lymphocytes to PHA was equal with that of uninfected controls.     

Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle

The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperm to penetrate at ovulation. We studied this mucus, its content of proteins and mucins, and the mucin O-glycosylation in cervical secretions before, during, and after ovulation. Cervical mucosal secretions from 12 subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, and stained with silver, Alcian blue, or Coomassie Blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nano-LC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF membranes, and the O-glycans were released by reductive beta-elimination and analyzed with capillary HPLC-MS and -MS/MS. At least 50 neutral, sialic acid-, and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol Core 2 structures and a relative decrease of NeuAc residues are typical for ovulation, and NeuAc-6GalNAcol and NeuAc-3Gal- epitopes are typical for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

Specific indication of hemoproteins in polyacrylamide gels using a double-staining process

Hemoproteins were revealed in polyacrylamide gels in the presence of sodium dodecyl sulfate by staining with different benzidine derivatives. When the protein samples were treated with either beta-mercaptoethanol or dithiothreitol, a significant decrease in peroxidase activity of the proteins possessing noncovalently bound heme led to diminished staining. However, when Coomassie blue R-250 staining followed the hemespecific stain it was observed that the hemoprotein bands stained more intensely than duplicate sample bands that had been stained only with the Coomassie blue R-250. This staining property allows the indication of hemoproteins in gels even after the peroxidase yield has been significantly depleted by reducing agents.     

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.     

Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens

Infection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.     

A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.

We have developed a highly sensitive stain for visualizing proteins in polyacrylamide gels. Our modification of the procedure for de Olmos' neural, cupric-silver stain is 100 times more sensitive than the conventional Coomassie blue stain (e.g., detection of 0.38 vs 38 ng/mm² of serum albumin), and is comparable to the sensitivity attained with an autoradiogram of ¹⁴C-methylated proteins following a 5-day exposure. This silver stain will be especially useful for analysis of patterns of proteins from tissue where attainment of the high specific activity of isotope labeling which is necessary to detect minor protein components is expensive, technically difficult or, as in humans, prohibited. In preliminary results with material such as unconcentrated cerebrospinal fluid, the silver stain revealed a complex pattern of proteins not visible with Coomassie blue.     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Immunoregulation in experimental schistosomiasis: in vitro induction and assay of spleen cell suppressor activity

Spleen cells from normal CBA/J mice or mice infected with Schistosoma mansoni were exposed for 48 to 72 hr to either concanavalin A (Con A), soluble egg antigen (SEA), or soluble worm antigenic preparation (SWAP), treated with mitomycin C to prevent further DNA synthesis, and admixed with either normal or sensitized syngeneic spleen cells exposed to a concentration gradient of phytohemagglutinin (PHA) or SEA, respectively. Both nonspecific (by Con A) and "antigen-specific" (by SEA and SWAP in infected mice only) induction of suppression was observed when using PHA-induced blastogenesis as the final assay. The number of mice with inducible splenic suppressive activity and the degree of PHA suppression induced by exposure to SEA appeared to decline between 8 and 20 weeks of infection. In contrast, when the response of spleen cells from mice infected for 8 weeks to SEA served as the final assay, strong suppressive activity was induced from the spleen cells of all chronically infected mice (20 weeks of infection). This model permits parallel analysis of the induction of suppressor activity by nonspecific and schistosome antigen-specific signals during the course of this chronic, immunoregulated condition, schistosomiasis mansoni.     

Simple, time-saving dye staining of proteins for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue

A fixation-free and fast protein-staining method for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol comprises staining and quick washing steps, which can be completed in 0.5 h. It has a sensitivity of 10 ng, comparable with that of conventional Coomassie Brilliant Blue G staining with phosphoric acid in the staining solution. In addition, the dye stain does not contain any amount of acid and methanol, such as phosphoric acid. Considering the speed, simplicity, and low cost, the dye stain may be of more practical value than other dye-based protein stains in routine proteomic research.