Adp

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...

Drw

0.1 M Sodium barbital buffer, pH 7.75 0.2 M NaCl 3.4 mM CaCl2 1.4 mM MgCl2 0.9 mM Pyroglutamyl-Gly-Arg-p-nitroanilide 3 Agarose solution 60 C Mix equal volumes of A and B solutions and pour the mixture onto a prewarmed glass plate to give a 2-mm-thick gel. Place an electrophorized SDS-PAG washed as described in Method 1 on top of a substrate-containing agarose gel and incubate at 37 C in a moist chamber for up to 12 h. Areas of UK activity appear as yellow bands in the agarose gel. Record the...

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...

Products that Cause a pH Change

Local acidic-alkaline pH change takes place in areas of acid electrophoretic gels where enzymes producing ammonia ions are localized. These enzymes are detected by incubation of electro-phorized gels in staining solutions containing the substrate and an appropriate pH indicator dye, e.g., Phenol Violet, which is light orange at acidic pH and becomes dark blue at alkaline pH. Examples are the detection of urease 3.5.1.5 UR, Method 1 , adenosine deaminase 3.5.4.4 ADA, Method 3 , and AMP deaminase...

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...

Subunit Structure

Transamidase, transglutaminase, fibrin stabilizing factor, coagulation factor XlIIa, factor XlIIa, fibrinoligase Protein glutamine alkylamine protein Ar5-alkyl glutamine NH3 see also General Notes Bacteria, fungi, plants, invertebrates, vertebrates Dimer vertebrates , see General Notes dabsylcadaverine-VIS dimethylcaseine in gel matrix A. 0.05 M Tris-HCl buffer, pH 7.5 0.2 mM Dabsylcadaverine 20 mM CaCl2 2 mM Dithiothreitol 14 U ml Thrombin B. 45 Ethanol in 10 acetic acid Incubate a 1 agarose...

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

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...

Multiple Replication of Electrophoretic Gels by Electroblotting

Electrophoretic transfer of enzymes from a single running gel onto immobilizing matrices e.g., Zeta Pore or Hybond-N membranes and Whatman DE 81 paper allows one to obtain multiple replicas, which can then be developed separately using different enzyme stains.36 It is important to remember when using this tactic that the faster-migrating enzymes should be detected on the first replica, while the slower-migrating ones should be on the last replica. Thus, use of the replication tactic requires...

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...