Catalogue Number: CYP207
Product Description: Sf9 insect cell microsomes containing human UGT1A4
Amount: 2.5 mg protein (0.5 ml)
Storage Buffer: 25 mM Tris-acetate (pH 7.6), 10% (v/v) glycerol, 1 mM EDTA
Storage: Store at -80°C. Avoid frequent temperature changes. Thaw on ice.
Intended Use: For laboratory (research) purposes only
Typical Batch Characteristics
Protein Concentration: 5 mg/ml
Typical UGT Activity Data
Substrate: Trifluoperazine (N-glucuronide formation)
Linear with time up to: 30 min
Linear with protein up to: 80 µg/ml
Vmax: 500 pmol/min/mg protein
Km (trifluoperazine): 20 – 30 µM
1. Data obtained from QC testing of UGT1A4 suggest that trifluoperazine N-glucuronide binds extensively to microsomal protein (although this has not been tested by us). Linear formation of product with respect to protein concentration is therefore only seen if control Sf9 insect cell microsomal protein is added to incubations to keep the total protein concentration constant. Failure to control for total protein concentration (or to correct for protein binding) results in a non-linear response (the calculated enzyme activity decreases as the protein concentration increases). This is likely to be an issue only when using trifluoperazine as the test substrate.
2. We recommend allowing stock solutions of trifluoperazine N-glucuronide to warm up to at least 4°C (if stored in a freezer) and mixing them thoroughly by vortexing before preparing standard curves, as the glucuronide also seems to bind to plasticware. We have found that stock solutions of the glucuronide in water/acetonitrile, 50/50 (v/v) yield more linear standard curves than stock solutions prepared in water.