Substrate solution

In actual practice the soluble component usually is injected into the substrate solution after the insoluble monolayer has been spread. The reason is that if one starts with the solution, the surface tension may be low enough that the monolayer will not spread easily. McGregor and Barnes have described a useful injection technique [265]. 

The LC/TOF instmment was designed specifically for use with the effluent flowing from LC columns, but it can be used also with static solutions. The initial problem with either of these inlets revolves around how to remove the solvent without affecting the substrate (solute) dissolved in it. Without this step, upon ionization, the large excess of ionized solvent molecules would make it difficult if not impossible to observe ions due only to the substrate. Combined inlet/ionization systems are ideal for this purpose. For example, dynamic fast-atom bombardment (FAB), plas-maspray, thermospray, atmospheric-pressure chemical ionization (APCI), and electrospray (ES)... 

Under suitable conditions, a stream of solvent containing a substrate (solute) dissolved in it can be broken up into a spray of fine droplets at atmospheric pressure (nebulized). 

The antibody solution (1.6x10 M) and substrate solutions with various concentration from 10 M to 10 M were mixed on a BSA-coated plate. The mixed solution of antibodies and substrates was allowed to stand for 1 day at room temperature, and then transported to the ELISA plates pre-coated with BSA-hapten and BSA blocking buffer. Absorbance at 405 nm for the resulting enzymatic hydrolysis product (p-nitrophenolate) by alkalinephosphatase of the second antibody was recorded on an Immuno-Mini NJ-2300 to determine the amount of antibody bound to BSA-hapten. 

Inhibitor assay A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37°C. Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. 

Chohnesterase-inhibiting pesticides (e g., organophosphate and carbamate pesticides) are detected by dipping the developed chromatogram in a solution of the enzyme chohnesterase followed by incubation for a short period. Then the plate is dipped in a substrate solution, e.g., 1-naphthyl acetate/fast blue salt B. In the presence of the active enzyme, 1-naphthyl acetate is hydrolyzed to 1-naphthol and acetic acid, and the 1-naphthol is coupled with fast blue salt B to form a violet-blue azo dye. The enzyme is inhibited by the pesticide zones, so the enzyme-substrate reaction does not occur pesticides are, therefore, detected as colorless zones on a violet-blue background [36]. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

Enzyme electrodes. Guilbault52 was the first to introduce enzyme electrodes. The bulb of a glass electrode was covered with a homogeneous enzyme-containing gel-like layer (e.g., urease in polyacrylamide) and the layer was protected with nylon gauze or Cellophane foil when placed in a substrate solution (e.g., urea) an enzymatic conversion took place via diffusion of substrate into the enzymatic layer. 

A typical procedure for setting up a preparative incubation is as follows the substrate solution with a minimal amount of organic solvent or solvent mixture is mixed with buffer (with necessary additives) in an Erlenmeyer flask. Microsomes or S9 is then added to the mixture, followed by addition of NADPH (or UDPGA) stock solution. The flask is incubated at 37 °C in a water bath with gentle shaking. The reaction kinetics in a large-volume incubation will be different from the 0.2-1.OmL incubation, so progress of the reaction should be monitored closely with a short HPLC-UV-MS method. 

The dehydrogenation catalyst is covered with a thin liquid film of the substrate solution so as to prevent the catalyst inside the liquid phase and the substrate vapor in the gas phase from direct gas-solid contact. Under boiling and refluxing conditions, vigorously formed... 

The analytical phase generally involves the use of very dilute solutions and a relatively high ratio of oxidant to substrate. Solutions of a concentration of 0.01 M to 0.001 M (in periodate ion) should be employed in an excess of two to three hundred percent (of oxidant) over the expected consumption, in order to elicit a valid value for the selective oxidation. This value can best be determined by timed measurements of the oxidant consumption, followed by the construction of a rate curve as previously described. If extensive overoxidation occurs, measures should be taken to minimize it, in order that the break in the curve may be recognized, and, thence, the true consumption of oxidant. After the reaction has, as far as possible, been brought under control, the analytical determination of certain simple reaction-products (such as total acid, formaldehyde, carbon dioxide, and ammonia) often aids in revealing what the reacting structures actually were. When possible, these values should be determined at timed intervals and be plotted as a rate curve. A very useful tool in this type of investigation, particularly when applied to carbohydrates, has been the polarimeter. With such preliminary information at hand, a structure can often be proposed, or the best conditions for a synthetic operation can be outlined. 

For three endo-D-galacturonanases, isolated from the culture filtrate of Coniothyrium diplodiella,114-116 that differed in the extent of degradation of sodium pectate, a 50% decrease of viscosity of the substrate solution corresponded to the splitting of 3, 4, and 10% of the glycosidic bonds, respectively. A similar difference was found between two endo-D-galacturonanases isolated from tomatoes117 one... 

Briefly, the membrane is incubated in the appropriate buffer for the chemiluminescence substrate then placed on a glass plate and a thin layer of substrate solution is pipetted onto the membrane to completely cover the surface and allowed to incubate for the required time (usually... 

Fig. 3 Generic FIIA system. A heterogeneous format is shown. The antibodies are immobilized in the immunoreactor. The analyte and the labeled Ag (in this case with an enzyme) are passed through the system and the competition step takes place. The flow of the substrate solution through the system allows the determination of the amount of bound labeled Ag, which is then detected and measured...

---