©Michael Court
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UGT activity screen by C14UDPGA-TLC
IMPORTANT - This assay uses a radiolabelled compound so the following precautions
and procedures must be followed:
a. Use isolated work space with fresh underpad to mop any (unlikely) spills
b. Wear lab coat and gloves
c. Label must be kept in locked refrigerator
d. Record amount of label used with each experiment in log book under refrigerator
e. Record any label disposed in sink on log next to sink
f. Clean work area after each experiment and dispose of any solid hot material by putting
into C14 waste container; hot liquids may be flushed down sink
g. Perform “wipe test” after each experiment and once each week while experiments are
being conducted
Prepare radiolabelled C14UDPGA cofactor mix
a. “Hot tube”:
i. Add sufficient C14UDPGA (dissolved in ethanol) to 1.5 mL screw cap
polypropylene tube
1. Generally 10 uL (25 uCi / mL) for each 100 uL incubation volume
ii. Dry down in vacuum oven at 450C (several hours)
b. “Cold tube”:
i. To a separate 1.5 mL tube add:
1. 0.645 mg UDPGA powder / 100 uL incubation
2. 10 uL 100 mM phosphate buffer pH 7.5 / 100 uL incubation
3. 10 uL 50 mM magnesium chloride / 100 uL incubation
4. Note: It’s a good idea to make up a little more to account for pipetting
loss and to use for the standard curve
c. Add the contents of the “cold tube” to the dried “hot tube”, cap and vortex.
d. Store solution on ice.
Prepare incubation tubes with desired substrate.
a. Dissolve substrate in methanol to 1 mM concentration (or 2x desired final
concentration)
b. Add 50 uL substrate in methanol (500 uM final concentration) to each incubation tube
i. Assumes 100 uL incubation volume
ii. Use 1.5 mL screw cap polypropylene tubes
c. Dry down in vacuum oven at 450C (up to 1 hr)
Prepare enzyme containing solutions
a. Prepare on ice
b. Liver (or other tissue) and expressed UGT microsomes
i. Use HLMs as positive control
ii. Use vector transfected (preferable) or untransfected cells as negative control
c. Dilute to 1 mg / mL with 50 mM phosphate buffer
d. To each incubation tube add either:
i. 1 uL Alamethicin (2.5 mg / mL methanol); 10 uL 50 mM saccharolactone in
water; 19 uL 50 mM phosphate buffer (make mastermix and add 30 uL)
ii. (OR just add 30 uL of 50 mM phosphate buffer)
e. Add 50 uL microsomes to each incubation tube
i. Important – ensure microsomes well mixed since they may settle
©Michael Court
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ii. Do not vortex recombinant UGTs – can cause denaturation
f. Mix by flicking tube
Incubation
a. Place incubation tubes into 370C water bath incubator
b. Start reaction by adding 20 uL of cofactor mix from “hot tube”
c. Cap tube
d. Incubate for desired time – up to 6 hours for quantitative data or overnight (16-18
hours) for qualitative activity screen
e. Stop reaction by adding 100 uL of ethanol
f. Centrifuge in microcentrifuge at max speed for 5 minutes
g. Transfer fixed amount (usually 190 uL) to new microcentrifuge tube
h. Dry down in vacuum oven
i. Redissolve by adding 30 uL 50% acetone in water and vortexing
j. Centrifuge for 30 – 60 seconds
Thin layer chromatography
a. Perform in fume hood
b. Fill TLC chamber with appropriate solvent mix and allow to saturate for several hours
to overnight
i. Original Bansal and Geissner mix (Use for all except N-glucuronides):
1. 1-Butanol – 140 mL
2. Acetone – 100 mL
3. Acetic acid – 18 mL
4. Water – 60 mL
5. Ammonium hydroxide – 1.5 mL
ii. Alkaline Bansal and Geissner mix (Use for N-glucuronides but NOT for
COOH-glucuronides):
1. 1-Butanol – 140 mL
2. Acetone – 140 mL
3. Water – 80 mL
4. Ammonium hydroxide – 40 mL
iii. Mackenzie mix (not N-glucuronides):
1. Toluene – 140 mL
2. Methanol – 60 mL
3. Acetic acid – 20 mL
iv. Radominska mix (not N-glucuronides):
1. Chloroform – 130 mL
2. Methanol – 50 mL
3. Acetic acid – 4 mL
4. Water – 8 mL
c. Ensure TLC chamber is completely sealed using thin film of silicone lube under lid
d. Apply sample to loading (thick) section of TLC lane
e. Allow samples to completely dry by placing plate sample side up in front section of
fume hood with door 90% closed and high exhaust turned on – creates drying breeze.
f. Develop plate by placing in TLC chamber (sample side at bottom – in the mobile
phase) and allowing solvent to travel almost up to the top of the plate.
©Michael Court
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g. Allow plate to completely dry by placing it sample side up in front section of fume
hood with door 90% closed and high exhaust turned on – creates drying breeze.
h. Once COMPLETELY dry wrap entirely in one sheet of plastic wrap
i. Tear off sheet of plastic wrap at least 2-3 times width of TLC plate and place on
bench
ii. Place TLC plate onto middle of plastic wrap with lane/sample side down
iii. Wrap plate completely – sample side should only have one layer of wrap
Expose plate using phosphorimager plate
a. Open phosphoimager cassette and blank the plate by placing on X-ray viewer box for at
least 20 minutes
b. Place TLC plate onto phosphorimager plate – lane/sample side down
c. Close cassette, label it and place it in drawer (dark place) for 24 – 72 hours
Read phosphorimager plate and quantitate percent of label conversion.