| Contributor: |
The Laboratory of Jasper Rine at the University of California, Berkeley
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| This protocol measures the ability of a yeast extract (made from the yeast strain of your choice) to phosphorylate Histone H1. Phosphorylation of Histone H1 is commonly seen as a measure of CLB2/CDC28 activity. |
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1. Use whole cell extracts from yeast strains of interest (see Protocol on Whole Cell Yeast Extract). Dilute yeast extract to a concentration of 1 mg/ml (in buffer used to make the extract or Assay Buffer).
2. Assemble the reaction in a microcentrifuge tube on ice:
200 μM ATP
100 μg/ml of Histone H1 protein
0.5 μCi of γ-[32P]-ATP (3000 Ci/mmol) (CAUTION! see Hint #1)
5 μg Yeast Extract
Make up in Assay Buffer (see Hint #2)
3. Incubate the tube at 30°C for 10 min.
4. Add 10 μl of 2X SDS-PAGE Sample Loading Buffer to tube to stop the kinase reaction.
5. Analyze the sample on a 15% SDS-Polyacrylamide gel and detect by autoradiography (see Protocol on SDS-PAGE).
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| SDS-PAGE Sample Loading Buffer (2X) |
| 2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT
0.04% (w/v) Bromophenol Blue
20% (v/v) Glycerol
125 mM Tris-Cl, pH 6.8
4% (w/v) SDS
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| Assay Buffer |
| 20 μg/ml Antipain
1 μg/ml Leupeptin
7.5 mM MgCl2
1 μg/ml Pepstatin
1 mM Phenylmethylsulfonyl Fluoride (PMSF)
20 mM Tris-Cl, pH 7.5
1 mM Benzamidine
10 μg/ml Aprotinin
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2-Mercaptoethanol
ATP
gamma-[32P]-ATP
Antipain
Histone H1 Protein
Pepstatin
Benzamidine
Glycerol
Bromophenol Blue
Tris
Leupeptin
SDS
PMSF
Magnesium Chloride
Aprotinin
DTT
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The total volume of the kinase reaction should be determined empirically.
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