- Fat bodies from 5 larvae were homogenized in 200 ul of 50 mM
potassium phosphate pH 7.5, 1 mM MgCl2, 0.2% NP40 using a
small blue pestle and a microfuge tube.
- Centrifuge the homogenate at for 5 minutes in the microfuge at 4 oC.
- Transfer the superntant to a new tube.
- Add 50 to 100 ul of extract to 1 ml of 50 mM potassium phosphate
pH 7.5, 1 mM MgCl2, 1 mM CPRG* in a disposable cuvette and
incubate at 37 oC. Set up a blank with buffer alone.
- Record the OD574 at 30 minute intervals for a period
of 3 hours.
- Measure total protein in an aliquot of the extract using the
Bio-Rad protein assay.
- Beta-galactosidase activity is defined as absorbance
units/hour/milligram protein.
*
CPRG = Chlorophenol red-beta-D-galactopyranoside