Reaktion #92786

Reaktionsgleichung

sodium acetate

p-nitrophenyl-α-D-glucopyranoside

glycine NaOH

→

pNPG

Reaktionsbedingungen

Aufarbeitung

1. 1
   Filtration30 μl filtered supernatant
2. 2
   workup.ADDITIONTo each well 50 μl NaAc buffer and 120 μl substrate was added
3. 3
   SonstigeThe reaction was terminated
4. 4
   workup.ADDITIONby adding 100 μl

Vorschrift

The reagent solutions were: NaAc buffer: 200 mM sodium acetate buffer pH 4.5; Substrate: 50 mM p-nitrophenyl-α-D-glucopyranoside (Sigma N-1377) in NaAc buffer (0.3 g/20 ml) and stop solution: 800 mM glycine-NaOH buffer pH 10. 30 μl filtered supernatant was placed in a fresh 96-well flat bottom MTP. To each well 50 μl NaAc buffer and 120 μl substrate was added and incubated for 30 minutes at 50° C. (Thermolab systems iEMS Incubator/shaker HT). The reaction was terminated by adding 100 μl stop solution. The absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 384 plus) and the activity was calculated using a molar extinction coefficient of 0.011 μM/cm.

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