Enzymes, selected staining

Enzyme staining Sometimes (e.g., with native gel electrophoreses), the activity of an enzyme survives the electrophoresis. Then the experimenter can try to selectively stain the enzyme bands in the gel using the enzyme activity. Proteases or enzymes that release phosphate or CO2 are well suited. 

R. Sen and C. M. Hepper, Characterization of vesicular-arbu.scular mycorrhizal fungi (Glomus spp.) by. selective enzyme staining following polyacrylamide gel electrophoresis. Soil Biol. Biochem. 18 29 (1996). 

The ability to select the relative size of the antibody—enzyme complex is important, depending on the assay application. Low-molecular-weight conjugates may be more optimal for immunohistochemical staining or blotting techniques where penetration... 

The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure-activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Exploration of these SAR studies were undertaken to optimize inhibitory activity and to improve the physicochemical properties of the sialic acid-based influenza virus sialidase inhibitor. A few in vitro assays are commonly employed to measure the effectiveness of influenza virus sialidase inhibitors. The first involves a fluorometric assay that measures release of a synthetic fluorophore following its cleavage from Neu5Ac by sialidase. Dye-uptake assay, such as the Neutral Red uptake assay, measures the uptake of a vital stain, Neutral Red in cell culture. The process requires intact membranes and active metabolism in the cell, and is expressed as percent protective rate against virus infection. The plaque-reduction assay is used to measure sialidase inhibition indirectly in cell culture, and provides some measure of the inhibitor s effect on the viability of the influenza virus. In vitro and in vivo systems for analysis of inhibitors of influenza virus enzymes have been reviewed.71... 

Because of these limitations, polymer-based immunohistochemical methods that do not rely on biotin have been introduced and are gaining popularity (5). These methods utilize a unique technology based on a polymer backbone to which multiple antibodies and enzyme molecules are conjugated. In the EPOS (Enhanced Polymer One Step) system, as many as 70 enzyme molecules and about 10 primary antibodies were conjugated to a dextran backbone. This allowed the entire immunohistochemical staining procedure, from primary antibody to enzyme, to be accomplished in a single step (6). On the other hand, one limitation of this method was its restriction to a select group of primary antibodies provided by the manufacturer, and not suitable for user-supplied primary antibodies. 

In cases where it is necessary to evaluate non-specific binding potentially caused by sources other than the primary antibody, additional patient tissue sections may be stained with selected reagents. For example, tissues may be stained with just the secondary antibody and/or the enzyme followed by application of the substrate/ chromogen. In cases where the suspected non-specific staining may be the result of endogenous enzyme present within the tissue, this can be confirmed by application of the substrate/enzyme only. 

More recently we have investigated whether the ubiquitin-proteosome system is perturbed in the heart of human DCM patients (Weekes et al, 2003). As in bovine DCM, expression of the enzyme UCH was elevated more than 8-fold at the protein level and elevated more than 5-fold at the mRNA level in human DCM. Moreover, this increased expression of UCH was shown by immuno-cytochemistry to be associated with the myocytes, which do not exhibit detectable staining in control hearts. Overall protein ubiquitination was increased 5-fold in DCM relative to control hearts. Using a selective affinity purification method we were able to demonstrate enhanced ubiquitination of a number of distinct proteins in DCM hearts. We have identified a number of these proteins by mass spectrometry. Interestingly many of these proteins were the same proteins previously found to be present at reduced abundance in DCM hearts (Corbett et al, 1998). This new evidence strengthens our hypothesis that inappropriate ubiquitin conjugation leads to proteolysis and depletion of certain proteins in the DCM heart and may contribute to loss of normal cellular function in the diseased heart. 

Chole- Relating to the biliary system, cholestasis The failure of the normal bile flow to the intestine, causing cholestatic jaundice, cholinergic Nerve fibres that release ACETYLCHOLINE, cholinesterases Enzymes that hydrolyse choline esters, especially ACETYLCHOLINE of which there are two main forms acetylcholinesterase ( true cholinesterase ) is specific for acetylcholine, rapid in this action, and has a discrete distribution being especially located near cholinergic nerve terminals (and in erythrocytes) butyrylcholinesterase ( pseudo cholinesterase) is less selective and is able to hydrolyse some drugs (e.g. SUCCINYLCHOLINE CHLORIDE). Many drugs are known that inhibit the action of these enzymes. See anticholinesterases. chromatin A protein found in the nucleus which stains with basic dyes. It is used in the study of the behaviour of... 

To detect the positions of electrophoretically separated DNA fragments, would ethidium bromide or a biotinylated DNA probe (used in conjunction with an avidin-enzyme conjugate and activity stain) be expected to provide positional data for all DNA fragments present in the sample Which would provide selective data for DNA fragments containing a particular sequence ... 

The choice for the multiple immunoenzymatic staining method is determined by several parameters (i) whether or not the same enzyme should be used for both staining procedures (ii) if unrelated enzymes are used, the selection of the second enzyme, in addition to the generally used POase (iii) the design of the two systems (direct conjugation, unlabeled method, bridge method, etc.) to avoid interference between them and, (iv) the species origin of the various antibodies. 

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