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The Laboratory of Donald Rio at the University of California, Berkeley
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1. Prepare kinase reaction as follows:
100 to 500 ng protein of protein substrate (see Hint #3)
1 μl of DNA-PK (HeLa Heparin 0.3 M pool)
1 μl of 10X DNA-PK Assay Buffer (50 mM final KCl concentration, see Hint #1)
1 μl of Excess DNA Solution
1 μl of γ-[32P]-ATP (10 μCi)
0.5 μl of 10 mM ATP
add ddH2O to give a final volume of 10 μl.
2. Incubate reaction mix at 30°C for 10 min.
3. Add 1 μl of 0.25 M EDTA.
4. Add 3 μl 4X SDS Sample Buffer.
5. Place tubes in boiling water bath for 2 to 3 min.
6. If transposase was used as the substrate, load samples onto a 7% polyacrylamide mini-gel. If KP protein was used, load the samples onto a 12% polyacrylamide mini-gel (see Protocol on Casting, Preparing and Running Polyacrylamide Mini-gels).
7. Stain gel with Coomassie blue and fix as usual.
8. Wash gel in ddH2O and dry on a gel dryer.
10. Perform autoradiography of gel using an X-ray intensifying screen.
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| 0.5 M DTT |
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| 3 M KCl |
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| 1 M MgCl2 |
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| 1 M HEPES-KOH, pH 7.6 |
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| SDS Sample Buffer (4X) |
| 40% (v/v) Glycerol
20% (v/v) 2-Mecraptoethanol
0.25 M 1 M Tris-HCl
8% (w/v) SDS
0.002%(w/v) Bromophenol Blue
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| 10 mM ATP |
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| Excess DNA Solution |
| 100 μg/ml DNA (sonicated calf thymus, oligonucleotides, or DNA restriction fragments)
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| 14.1 M 2-Mercaptoethanol |
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| γ-[32P]-ATP |
| 6000 Ci/mmole γ-[32P]-ATP (10 mCi/ml; CAUTION! see Hint #2)
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| 1 M Tris-HCl, pH 6.8 |
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| DNA-PK Assay Buffer |
| 10 mM DTT
0.5 M KCl (see Hint #1)
50 mM MgCl2
250 mM HEPES
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| 0.25 M EDTA |
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Bromophenol Blue
DNA (sonicated calf thymus, oligonucleotides or DNA restriction fragments)
Potassium Hydroxide
Purified DNA-dependent Protein Kinase
SDS
Tris
HEPES
Magnesium Chloride
2-Mercaptoethanol
EDTA
Potassium Chloride
DTT
gamma-[32P]-ATP
ATP
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1. If your protein solution is in a high salt, you will need to make up the 10X Assay Buffer that will adjust the final concentration of KCl to 50 mM.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. The substrate can be either protein or peptide and can be either a free solution or immobilized on GST or NTA-agarose beads.
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2. Finnie NJ, Gottlieb TM, Blunt T, Jeggo PA, Jackson SP. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc. Natl. Acad. Sci. USA 1995 Jan 3;92(1):320-324.
1. Anderson CW, Lees-Miller SP. The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot. Gene Expr. 1992;2(4):283-314.
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