Standard enzymatic activity assays were performed using pNP-β-GlcNAc (Sigma, USA) and pNP-β-GalNAc (Sigma, USA) as a substrate. The assays were performed at a substrate concentration of 5 mM in McIlvaine buffer, pH 4.0–6.0 at 30°C for Drosophila FDL and at 37°C for the other hexosaminidases. The total reaction volume was 40 µL and incubation time was 2 h. All purified enzymes were tested for additional activities using a range of p-nitrophenyl-sugars as substrates at a concentration of 5 mM for 4 h. After the addition of 200 µL of 0.4 M glycine/NaOH pH 10.4, A405nm was determined on a Tecan Infinite M200 microtiter plate reader (Tecan, Austria). All enzymes were appropriately diluted for measurements in order to measure reactions within the linear range of the assay. Enzyme units are given in µmol product (nitrophenol) per minute. Specific activity is defined as units per milligram purified protein.
Pnp β glcnac
Manufactured by Merck Group
Sourced in United States
PNP-β-GlcNAc is a laboratory equipment product. It is a synthetic compound used as a substrate for the enzymatic detection and quantification of glycosidases, specifically β-N-acetylglucosaminidase. The product provides a colorimetric or fluorometric readout upon enzyme-catalyzed hydrolysis.
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Lab products found in correlation
2 protocols using pnp β glcnac
1
Enzymatic Assays for Hexosaminidases
2
Enzymatic Activities of Glycosyl Hydrolases
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The enzymatic activities of Hexes and chitinases measured at 25°C using 4-nitrophenyl-N-acetyl-β-acetylglucosamine (pNP-β-GlcNAc, Sigma-Aldrich). OfHex1, SmChb and TvHex were assayed in 20 mM sodium phosphate buffer (pH 6.0) and HsHex, CeHex and SpHex were assayed in 20 mM sodium citrate buffer (pH 4.5). After incubating for appropriate time, 0.5 M Na2CO3 was added to the reaction mixture and the absorbance at 405 nm was monitored using a Sunrise microplate reader (TECAN, Shanghai, China). As for Ki value determination, three substrate concentrations (0.075, 0.125 and 0.2 mM) were used. The concentrations of inhibitor varied for different enzyme. The Ki values and types of inhibition were determined by linear fitting of data in Dixon plots.
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