Enzyme specific staining

Figure 1. PAGE of purified rat liver xanthine oxidase. (A) Coomassie blue staining. (B) Enzyme specific stain. (C) Coomassie blue stain after Sephacryl S-300 showing protein contamination (arrow).

Proteinase-containing samples are incubated with a variety of class- or enzyme-specific proteinase inhibitors, separated on a polyacrylamide gel, and activity stained as described in Basic Protocol 3. A clear zone will be evident in lanes where the proteinase is active (i.e., in the absence of inhibitor or in the presence of a mismatched inhibitor). This clear zone will be absent in the lane containing the properly matched proteinase inhibitor, which provides information about the class or type of proteinase detected in the band. 

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. 

Enzyme histochemical staining of muscle fibers can identify abnormal levels of respiratory Complexes II, IV, and V, while specific immunohistochemical stains can be used to... 

The location of individual proteins or protein subunits has been established by analyzing pure proteins and immunoprecipitates prepared using specific antisera, or by co-electrophoresis. Proteins containing cysteine residues can be specifically labeled using [ I]iodoacetamide and can be located autoradiographically. If analysis is performed under nondenaturing conditions, specific stains are available to locate certain types of protein, e.g., glycoproteins (A14, H12) and enzymes. 

The importance of preserving enzymes has been discussed earlier in this chapter. The activity of enzymes is usually measured by performing an assay specific to the enzyme being tested. It is also recommended to conduct washing tests over an extended time period on enzyme-sensitive stained fabrics and determine the extent of enzyme loss as a function of time. 

Gels are stained for proteins with oomassie brilliant blue R 250 by the usual procedure. Alternatively, gels can be stained for enzyme activity using specific staining methods. 

Using a novel antibody raised against a C-terminal peptide of the rat Pi subunit of soluble guanyiate cyclase, the enzyme is primarily localised to the vasculature rather than to cardiomyocytes with a switch of soluble guanyiate cyclase-specific staining fi om arterial smooth muscle cells to endotheUal cells during development (Mietens et al. 2001). In rat adult cardiomyocytes, soluble guanyiate cyclase expression was clearly detectable, but low in comparison to whole heart. 

Monoesterquats based on dimethylethanolamine [94] have been suggested in a number of patent applications for use in granular detergents, in the presence of other additives like anionic, nonionic, or zwitterionic surfactants, enzymes, various soil release polymers, and hydrophobic bleach additives. These compositions showed improved detergency on specific stains [82,95-101]. Esterquat-containing formulations for detergent compositions (powder, liquid, or tablet) are described [102]. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

Abstract Fluorescent molecules have been widely used as biomolecular labels, enzyme substrates, environmental indicators, and cellular stains and thus constitute indispensable tools in chemistry, physics, biology, and medicinal sciences. The large variation in the photophysics of the available fluorophores connected with chemical alterations give fluorescent probe techniques an almost unlimited scope for the detection of specific molecules and the investigation of intermolecular interactions on a molecular scale. 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

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