Atomic force microscopy of Mammalian urothelial surface.
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The mammalian urothelium apical surface plays important roles in bladder physiology and diseases, and it provides a unique morphology for ultrastructural studies. Atomic force microscopy (AFM) is an emerging tool for studying the architecture and dynamic properties of biomolecular structures under near-physiological conditions. However, AFM imaging of soft tissues remains a challenge because of the lack of efficient methods for sample stabilization. Using a porous nitrocellulose membrane as the support, we were able to immobilize large pieces of soft mouse bladder tissue, thus enabling us to carry out the first AFM investigation of the mouse urothelial surface. The submicrometer-resolution AFM images revealed many details of the surface features, including the geometry of the urothelial plaques that cover the entire surface and the membrane interdigitation at the cell borders. This interdigitation creates a membrane zipper, likely contributing to the barrier function of the urothelium. In addition, we were able to image the intracellular bacterial communities of type 1-fimbriated bacteria grown between the intermediate filament bundles of the umbrella cells, shedding light on the bacterial colonization of the urothelium. (+info)
Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting.
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We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion. (+info)
A membrane capture assay for lipid kinase activity.
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Phosphoinositide kinases such as PI3-kinase synthesize lipid second messengers that control diverse cellular processes. Recently, these enzymes have emerged as an important class of drug targets, and there is significant interest in discovering new lipid kinase inhibitors. We describe here a procedure for the high-throughput determination of lipid kinase inhibitor IC50 values. This assay exploits the fact that phosphoinositides, but not nucleotides such as ATP, bind irreversibly to nitrocellulose membranes. As a result, the radiolabeled lipids from a kinase assay can be isolated by spotting the crude reaction on a nitrocellulose membrane and then washing. We show that diverse phosphoinositide kinases can be assayed using this approach and outline how to perform the assay in 96-well plates. We also describe a MATLAB script that automates the data analysis. The complete procedure requires 3-4 h. (+info)
The use of dacron plates for dot enzyme-linked immunosorbent assay (dot-ELISA).
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Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Titration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparison with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 degrees C, 28 degrees C and -20 degrees C for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 degrees C. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate. (+info)
A nanogram-level colloidal gold single reagent quantitative protein assay.
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Effectiveness of surface protection for glass-ionomer, resin-modified glass-ionomer and polyacid-modified composite resins.
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The purpose of this study was to evaluate the effectiveness of several surface protectors for a glass-ionomer, a resin-modified glass-ionomer, and a polyacid-modified resin cement by determining dye uptake spectrophotometrically. 378 samples, made up of Ionofil U, Vitremer, and Dyract, were prepared and divided into groups of seven each. Positive and negative control specimens remained unprotected while the experimental specimens were protected with Finishing Gloss, Protect-It, LC Varnish, Adper Single Bond, or a nail varnish. The experimental groups and positive controls were immersed in 0.05% methylene blue solution, while the negative controls were immersed in deionized water. Results were evaluated using variance analysis. Of the Ionofil U group, Adper Single Bond exhibited the least effective surface coating among the materials tested, while the best surface protection was obtained with LC Varnish in the Dyract group. However, no statistically significant differences were observed in the Vitremer group. (+info)