0-Galactosidase reaction

Incubate at room temperature. Peroxidase reactions take about 5—30 mm to develop P-galactosidase reactions can take longer Judge the reaction time by eye (see Notes 4 and 5). Reactions may be stopped by adding 100 pL of appropriate stop solution to each well (5% SDS for peroxidase, lMNa2C03 for P-galactosidase). The SDS also solubilizes any precipitated products formed in the HRP reaction. 

If the ELISA color development takes more than a few minutes, continue the incubation m the dark p-galactosidase reactions tend to have a lower spontaneous background than peroxidase reactions, but take longer to develop, p-galac-tosidase reactions can be sped up by incubation at 37°C. 

The enzyme /3-galactosidase allows lactose metabolism in Escherichia coli. As shown in Figure 7-1, this enzyme catalyzes the hydrolysis of lactose into its monosaccharide units, galactose and glucose. A side product of the /3-galactosidase reaction, allolactose, is known to control the expression of the enzyme at the level of transcription. 

Use the following equation to determine the initial velocity of each /3-galactosidase reaction (micromoles of ONPG hydrolyzed per minute) ... 

Figure 31.1. Following the P-Galactosidase Reaction. The galactoside substrate X-Gal produces a colored product on cleavage by P-galactosidase. The appearance of this colored product provides a convenient means for monitoring the amount of the enzyme both in vitro and in vivo.

Electrochemical Microflow Systems, Fig. 4 Galactosidase reaction monitored by fluorescence in a nano-channel reactor (Redrawn from [58])... 

Procedures of the beta-galactosidase activity measuring using colour reaction with ONPG and X-Gal without cells permeabilization were developed and the detection limit at the level of 4 ppb has been achieved. The influence of the foreign ions (phosphate, sulphate, carbonate et. al) was studied. 

The rapid inactivation of human lysosomal / -D-galactosidase is noteworthy, as the reaction had to be carried out at 2 pH units above its pH optimum (pH 4.0) in order to minimize the spontaneous decomposition of the inhibitor. Similar conditions had to be employed for ) -D-xylosidase from Penicil-lium wortmamii (optimum at pH 3.5), which was studied at pH 7.0. 

P 19] Two micro syringes were filled with 0.32 mM p-nitrophenyl-y9-D-galacto-pyranoside in phosphate buffer (pH 8) and /3-galactosidase (20 U) in 10 ml of the same buffer [26]. Both solutions were pumped into the micro channel at the same flow rate (a few pi min ). The reaction was carried out for 0-30 min at 37 °C using a hot-plate. The residence time was set by adjusting the flow rate. After passing the micro channel, the reaction mixture was dropped into hot water to inactivate the enzyme. 

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