Reaction

SUBUNIT STRUCTURE Unknown or no data available Method 0.1 M Tris-HCl buffer, pH 8.8 60 mM Indole-3-acetaldehyde 5 mM NAD 20 Sucrose Cover the gel with staining solution and incubate in a humid chamber at room temperature. View the gel under long-wave UV light for fluorescent bands. Record the zymogram or photograph using a yellow filter. Notes This is the original method developed for acetate cellulose gel. It can also be applied to PAG and starch gel. The addition of PMS and MTT (or NBT) to...

Mdpcmt

I MDPCMT I STMe- HR-cysteine (in vitro) Stage 1. Incubate 200 J,l of cell or tissue extract prepared using 50 mM Tris-HCl (pH 8.3), 0.5 mM EDTA, and 1 mM dithiothreitol buffer with 10 J,l of Me-3H -guanine DNA substrate (200 to 500 dpm l) at 37 C for 90 min (although the majority of the repair reaction occurs within the first 5 min, the longer period of incubation is used to ensure completeness of reaction). Stage 2. Subject 30 J,l of the enzyme reaction mixture containing the radiolabeled...

Cellulose Acetate

Only continuous buffer systems are suitable for electrophoresis in cellulose acetate gel. The gel and the electrode buffers are usually of the same ionic strength. 10 mM phosphate (sodium dibasic) 2.5 mM citric acid Modifications There are numerous modifications of this buffer concerning the pH value (varies from pH 5.0 to 7.5) and final concentrations of phosphate (varies from 5 to 100 mM) and citrate (varies from 1.5 to 35 mM) constituents. 20 mM phosphate (sodium dibasic), adjusted to pH 7.0...

Edta

Salicylic acid Tb3+ EDTAUV (ternary luminescent lanthanide chelate complex) 1 mM Salicylaldehyde (dissolved in ethylene glycol Apply substrate solution A to the gel surface with a filter paper or 1 agarose overlay. Incubate the gel with application at 37 C for 30 min. Remove the first application and apply the next one containing developing solution (one part of solution B, one part of solution C, and three parts of deionized water). Observe luminescent XOX bands under 300- to 400-nm UV light....

Chromogenic Reactions

Chromogenic reactions are those that result in formation of a chromophore at sites of enzyme activities. The great majority of these reactions were adopted from well-tried histochemical or colorimetric enzyme assay methods. In a simple one-step chro-mogenic reaction a colorless substrate is enzymatically converted into a colored product. In a broad sense, the class of chromogenic reactions includes any reactions or set of reactions that reveal discrete zones (or bands) of enzyme activity...

Adp

Glutamylcysteine Ligase

Calcium phosphate (white precipitate) 0.14 M Tris-HCl buffer, pH 8.0 0.14 M MgCl2 14 mM L-Glutamate 14 mM L-a-Aminobutyrate 14 mM ATP Incubate an electrophorized PAG in the staining solution at 37 C until white bands of calcium phosphate precipitation appear. Store the stained gel in 50 mM glycine-KOH buffer (pH 10.0), containing 5 mM Ca2+, either at 5 C or at room temperature in the presence of an antibacterial agent. Notes Calcium phosphate precipitate may be counterstained with Alizarin Red...

Modes of Application of Staining Solutions

Staining solutions can be applied directly to the surface of elec-trophoretic gels or used as filter paper or agar overlays. A standard staining solution method is the earliest and the most widely used. It consists of placing an electrophoretic gel or gel slice in a special staining tray, adding the staining solution until the gel is completely covered by fluid, and incubating the gel at room temperature or 37 C usually in the dark until enzyme activity bands are visible. Specifically designed...

NADH and NADPH

Positive fluorescent zymograms may be obtained for almost all NAD P -dependent dehydrogenases that generate NAD P H from NAD P . These dehydrogenases are usually detected using the tetrazolium method, which is more convenient because it results in the development of dehydrogenase activity bands visible in daylight see Products Reducing Tetrazolium Salts in Section 1 . However, positive fluorescent stains are less expensive than tetrazolium stains because they do not require PMS and a...

Modes of Enhancement of Staining Intensity of Enzyme Activity Bands

These modes can be divided into two main groups. The first group is represented by methods that are applicable before the procedure of detection of enzyme activity bands. It includes methods of protection and activation of enzymes during preparation of enzyme-containing samples and their electrophoretic run. Some modifications of the sample application procedure can increase the sample amount applied onto the gel, and thus increase the enzyme amount in the gel and the staining intensity of...

References For Part Ii

Seligman, A.M. and Rutenberg, A.M., The histochemical demonstration of succinic dehydrogenase, Science, 113, 317, 1951. 2. Markert, C.L. and M ller, F., Multiple forms of enzymes tissue, ontogenetic, and species specific patterns, Proc. Natl. Acad. Sci. U.S.A., 45, 753, 1959. 3. Pearse, A.G.E., Histochemistry Theoretical and Applied, Vol. I, Williams amp Wilkins, Baltimore, 1968. 4. Burstone, M.S., Enzyme Histochemistry, Academic Press, New York, 1962. 5. Hunter, R.L. and Markert, C.L.,...

Products Reducing Tetrazolium Salts

The first histochemical method for the detection of enzyme activity using a tetrazolium salt was developed in 1951 by Seligman and Rutenberg.1 Markert and M ller2 were the first to adopt this his-tochemical procedure for detection of NAD P -dependent dehydrogenases on electrophoretic gels. These dehydrogenases produce reduced NADH or NADPH, the electron donors for reduction of tetrazolium salts, which are especially good electron acceptors. Reduction of a tetrazolium salt results in the...

Enzyme Source Subunit Structure

Diazonium Bushes

Citrulline phosphorylase, ornithine transcarbamylase Carbamoyl phosphate L-ornithine orthophosphate L-citrulline Bacteria, fungi, plants, vertebrates Trimer bacteria, fungi, plants, vertebrates A. 0.27 M Triethanolamine buffer, pH 7.5 5 mM L-Ornithine B. 20 mM Ammonium molybdate 0.5 Nitric acid Incubate the electrophorized gel in solution A at 37 C for 10 min. Rinse the gel in distilled water and place in solution B for 5 min. Rinse the gel again in water and immerse in solution C. Blue bands...

G3pd

A. 50 mM HEPES N-2-hydroxyethylpiperazine-W-2- ethanesulfonate buffer, pH 8.0 0.5 mM EDTA 4 mM Mg CH3COO 2 2 mM Pyrophosphate 0.2 U ml Aldolase ALD 0.2 U ml Triose-phosphate isomerase TPI 0.2 U ml Glycerol-3-phosphate dehydrogenase G-3-PD 100 nM D-Fructose-2,6-bisphosphate Mix equal volumes of A and B components of the staining solution and pour the mixture over the surface of the gel. Incubate the gel at 37 C and monitor under long-wave UV light. Dark nonfluorescent bands on a light...

Mtt

Mtt Nadph Reaction

100 mM Phosphate buffer, pH 7.6 5 mg ml Sucrose 1 U ml Glucose-6-phosphate dehydrogenase G-6-PD Apply staining solution as a 1 agarose overlay and incubate the gel at 37 C in the dark until dark blue bands appear. Fix the stained gel or agarose overlay in 25 ethanol. Notes This method is specific for SP. A dimeric SP was found in Pseudomonas saccharophila,3 while the enzyme from Leuconostoc mesenteroides proved to be a monomer.4 Gabriel, O. and Wang, S.-F., Determination of enzymatic activity...

Recording and Preservation of Zymograms

There are at least three different methods of recording the enzyme patterns developed on electrophoretic gels 1 schematic recording of zymograms, 2 photography of zymograms, and 3 the tracing of the band position on paper overlays or cellulose acetate gels. After recording, zymograms can be stored in special fixative solutions or dried. Schematic recording of zymograms involves a two-dimensional representation of the banding patterns observed on developed gels. Although such representation...

Specificity of Zymogram Methods and Some Related Problems

Most of the zymogram methods presented in this part are enzyme specific. However, some methods can detect more than one enzyme. This results from the ability of some enzymes to utilize one or more of the applied reagents, or to use certain buffer constituents coupled with the applied staining solution reagents. Another reason for the development of unexpected enzymatic bands is the combined effect of two comigrating endogenous enzymes, one of which acts as a linking enzyme for the other in the...

Products Capable of Coupling with Diazonium Salts

Many hydrolytic enzymes e.g., aminopeptidases, esterases, gly-cosidases, phosphatases are visualized on electrophoretic gels using artificial substrates that are naphthol or naphthylamine derivatives. When naphthol or naphthylamine is liberated enzy-matically, it immediately couples with a diazonium salt. An insoluble colored precipitate an azo dye is formed as a result of the coupling reaction in gel areas where specific hydrolase activity is located. There are two main components of the azo...

Preparation of Staining Solution

Each staining solution is buffered with the staining buffer at a specific pH. The pH value of the staining buffer used for the enzyme stain is a compromise between 1 the pH optimum of the enzyme activity, 2 the pH optimum of the staining reaction used in the detection method, 3 the pH value of the buffer used for preparation of the gel, and 4 the pH optimum of the linking enzymes, if those are involved in staining reaction s . Many substrates e.g., those used by dehydrogenases are acids. In...

Products that Influence the Starch Iodine Reaction

The starch-iodine chromogenic reaction has been known for more than a century. However, iodine I2 is insoluble in water. Its solubility increases considerably in the presence of iodide I- , so KI is usually included in iodine solutions. Some compounds influence the starch-iodine reaction due to their ability to reduce iodine to iodide, which fails to react with starch. Such reductive properties toward iodine are displayed by some compounds bearing free thiol groups, e.g., by reduced...