CPRG Assays on Crude Extract of Adult Flies
(Modified by DSG, 3/15/00) Needed
Homogenization Buffer: 50 mM Potassium Phosphate (pH 8), 1 mM MgCl₂

To make 100 ml of 1 M Potassium Phosphate (pH 8) as described in Maniatas, combine 94  ml 1 M K₂HPO₄ and 6 ml 1 M KH₂PO₄

CPRG Assay solution: 1 mM CPRG, 50 mM Potassium Phosphate (pH 8), 1 mM MgCl₂

50 mM CPRG stock, made up in H₂O, stored at -20 ^oC  (30 mg CPRG/ml)
CPRG = Chlorophenol red-beta-D-galactopyranoside (available from Roche)

Bio-Rad Protein Assay solution (Dye Reagent)

Stored at 4 ^oC in fridge by Hokuto's bench

Additional Information

• This assay should be conducted on heterozygous flies to insure that each line contains one copy of the insert. To obtain heterozygotes, mate virgin transformed females with yw males (or visa versa). Select progeny with colored eyes.

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Procedure Heat Shock Conditions

1. Briefly anesthetize flies with CO₂ and transfer 5 to 10 flies into a 1.7 ml microfuge tube that has 4 holes punched in the lid with a 20 gauge needle. Try to use microfuge tubes that are compatible with the small blue homogenization pestles.  At present (3/15/00), we have blue colored tubes from Kontes that work well.  The pestle should reach the bottom of the tube.  Also, use the same number and sex of flies when comparing different lines.  Note that in males, genes on the X chromosome are likely to be expressed at twice the level found in females.
2. Allow the flies to recover for 20 minutes at room temperature before heat shocking the flies.
3. Heat shock the flies by floating the tubes in a 37 ^oC water bath for 1 hour.  The water bath provides high humidity so the flies are less likely to dehydrate.  None of the flies should die and they will survive heat shocks of at least 2 hours under these conditions.
4. After heat shock, place each tube of flies on dry ice.

Non-Heat Shock Conditions.

1. While putting flies into tubes for heat shocks, put another group of flies in a separate tube.  Place the nonheat shocked flies on dry ice.

Prepare Crude Extract of Adult Flies.

1. Add 100 ul Homogenization Buffer and place the tube on ice.
2. Grind the flies for approximately 30 seconds on the Wheaton Overhead Stirrer. Use a small blue pestle. Set the motor at 3.3. Keeping the tube at a slight angle, move it up and down against the pestle to grind the fly tissues. Make sure to dislodge the "plug" of fly tissue with a pipette if the fly tissue becomes jammed into the bottom of the tube by the pestle. Turn the motor off before you withdraw the pestle to avoid splattering. Also, when removing the pestle, wipe the tip against the side of the tube. Rinse the pestle with ddH₂O and wipe dry with a clean paper towel in between samples. Freeze each sample on dry ice after each has been homogenized.  This should help lyse cells and maintain the consistency of the samples if the homogenization takes some time to complete.
3. Leave all the samples on dry ice for at least 10 minutes.  Then add 900 ul of homogenization buffer, thaw, and mix the samples.
4. Shake samples in the benchtop shaker for 1 minute at room temperature.
5. Microfuge the samples 2 minutes at maximum speed.
6. Transfer 100 ul of each clear fly lysate to a fresh tube. Avoid transferring insoluble material. Place the tubes on ice.

CPRG Assay

1. For each sample, put 1 ml of the CRPG Assay Solution into a cuvette.
2. Transfer 20 or 50 ul of the clear fly lysate into the cuvette. For the wild-type promoter, the absorbance can exceed 1 after a 2 hour incubation.  Try to avoid transferring any solid material. Mix by pipetting up and down.
3. Set up a blank cuvette with 1 ml CPRG Assay Solution and 20 or 50 ul of Homogenization Buffer.
4. Incubate the samples at 37 ^oC.
5. Read the OD₅₇₄ after 1, 2, 4 and 24 hours, using the blank to calibrate the spectrophotometer at each time point. Color production is linear for approximately 8 hours.

Bio-Rad Protein Assay
You should normalize your measurements for differences in protein concentrations. To determine the protein concentration of each extract, use the Bio-Rad Protein Assay. To determine absolute protein concentrations, you will need to set up a standard curve with known amounts of BSA. However, it should be sufficient to express your ß-galactosidase measurements as OD units of ß-gal /hr /OD units of protein. 20 ul of lysate from 5 males will give a reading of about 0.2 to 0.25.

1. For each sample, add 800 ul of ddH₂O to a clean cuvette.
2. Add 200 ul of "Dye Reagent Concentrate". Pipette up and down to mix.
3. Add 20 ul of the crude fly extract. Pipette up and down to mix.
4. Set up a blank with water, the Dye Reagent and 20 ul Homogenization buffer.
5. Measure the OD₅₉₅, using the blank to calibrate the spectrophotometer.