BSH Activity Assays.

BSH specific activity assay (by ninhydrin assay) 

1.    Enzyme assay (50uL reactions) carried out in 50 mM NaPhos pH 6.0, 10 mM DTT at 37C containing 9 mM BA and 1-10 nM BSH.

2.    Quench reaction with 50uL of 15% trichloroacetic acid. 

3.    Centrifuge at RT 12,000 x G for 2 minutes to pellet precipitate.

4.     Add 25uL of quenched reaction to 475uL of ninhydrin reaction (250uL of 1% ninhydrin in    0.5M Na-Citrate (pH 5.5), 600uL of glycerol, 100uL of 0.5M Na-Citrate (pH 5.5)). 

5.     Boil samples for 14 min and cool to room temperature.

6.     Aliquot reactions 200uL per 96 clear well and read at 570nm. 

7.     Include a standard curve of glycine or taurine for each assay.