| Contributor: |
The Laboratory of Donald Rio at the University of California, Berkeley
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| CAT activity can be used as a reporter to measure transcription. The CAT gene is inserted downstream from a promoter of interest and this construct is introduced into cells for expression. Extracts are prepared from these cells and CAT activity results from promoter-driven transcription. |
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1. Combine the following components:
Up to 50 μl of cellular extract (see Hint #2)
Bring the final volume to 90 μl using 0.25 M Tris Buffer (see Hint #3).
2. To the 90 μl reaction add the following:
1 μl of Labeled Chloramphenicol (CAUTION! see Hints #1 and #4)
1 μl of n-Butyryl Co A
8 μl of 0.25 M Tris Buffer
The final reaction volume should be 100 μl.
3. Incubate at 37°C (see Hint #5).
4. Terminate the reaction by adding 200 μl (2 volumes) of Pristine/Xylene Solution.
5. Mix well by inversion, centrifuge to separate the phases, and save approximately 90% of the organic phase (180 μl).
6. Measure the radioactivity in scintillation counter using an organic scintillant.
7. Calculate the conversion by comparing cpm of positive CAT (no cellular extract) control and negative control (no cellular extract) with cpm of cellular extract.
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| n-Butyryl Co A |
| Prepared in ddH2O
50 mM n-Butyryl Coenzyme A
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| 0.25 M Tris Buffer |
| 0.25 M Tris-HCl, pH 7.8
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| Pristine/Xylene Solution |
| 2 parts Pristine
1 part Xylene
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| TE Buffer |
| 1 mM EDTA
10 mM Tris-HCl, pH 7.5
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| Labeled Chloramphenicol |
| Prepared in TE buffer
0.2 mCi [14C]-Chloramphenicol (approximately 54 Ci/mmol) (CAUTION! see Hints #1 and #4)
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| Pure Chloramphenicol (CAT) |
| Prepared in TE Buffer
2.5 Units Chloramphenicol Acetyl Transferase per μl Chloramphenicol
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[14]C-Chloramphenicol
EDTA
Scintillation Fluid
n-Butyryl Coenzyme A
Xylene
Chloramphenicol
Pristine
Tris
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The volume of cellular extract should be determined empirically and should not exceed 50 μl.
3. Remember to prepare one reaction without cellular extract as negative control. Also, remember to prepare one reaction without cellular extract and with pure CAT as positive (100% conversion) control.
4. Labeled Chloramphenicol (14C or 3H) should be lyophilized (without heat) if in Ethanol, then resuspended in TE Buffer to a concentration of 0.2 mCi/ml CAM. Purify Chloramphenicol by adding an equal volume of Xylene, centrifuge to separate phases, save the aqueous phase. Repeat Xylene extraction (allow the organics to evaporate in hood).
5. The time of incubation should be determined empirically and is usually greater than 0.5 hr.
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