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Assay Method Information

Assay Name:  Kinase Assay
Description:  ALK-5 kinase assay methods have been described in the art (see e.g., Laping et al. (2002) Mol. Pharmacol. 2002; 62: 58-62). The compounds named in the specified Examples were tested as follows for inhibition of ALK-5 autophosphorylation activity and of the ALK-5 phosphorylation of α-Casein.Materials: Buffer. 50 mM HEPES, pH 7.6, with 10 mM NaCl, 10 mM MgCl2, and 1 mM DTT. GST-ALK-5 protein 0.44 mg/ml (roughly 7 μM stock). A 1:350 dilution gives a 20 nM stock, which translates to 2 nM final in assay. Human ALK-5 was expressed in Sf9 insect cells infected with Baculovirus expressing a ALK-5 truncation sequence (amino acids H149-M503), fused at the N-terminus to Glutathione S-transferase GST, in a pFastBac vector (Invitrogen). The cells were disrupted by sonication at 4° C. The lysate was centrifuged at 40,000×g for 45 minutes, and the supernatant applied to a 10 ml column of Glutathione Sepharose 4 Fast Flow (Amersham Bioscienses) equilibrated with 100 mM Tris-HCl pH 7.6 buffer containing 300 mM NaCl, 10% glycerol, 1% NP40, 2 mM dithiothreitol (DTT) and one Protease Inhibitor complete EDTA-free tablet per 50 ml (Roche). The column was washed with 5 column volumes of 50 mM Tris HCl pH 8.0 containing 150 mN NaCl, 10% glycerol, 2 mM DTT and one Protease Inhibitor complete EDTA-free tablet per 100 ml. The column was eluted with wash buffer containing 8 mM reduced glutathione. Fractions were collected and dialyzed overnight in 20 mM Tris HCl pH 8.0 containing 10% glycerol, 150 mM NaCl, 2 mM DTT and 1 mM 4-(2-aminoethyl)-bezenesulfonylfluoride.HCl (AEBSF) (Sigma) at 4° C. α-Casein (Sigma, #C8032) is made up at 2 mM in Buffer (50 mg/ml). Cold ATP contains 10 μM cold ATP (from a 10 mM stock in Buffer). Hot ATP consists of 0.5 μCi/well □-33P-ATP (Amersham, AH9968) in Buffer. Assay Buffer Per 10 ml Buffer 1 ml of 500 mM HEPES (pH 7.6) 20 μl of 5 M NaCl 100 μl of 1 M MgCl2 10 μl of 1 M DTT (dithiothreitol) Assay Method:In a 96 well filter-bottom plate (Millipore, #MSDV N6B 50), 58 μl Assay Buffer is added to reach well. Add 10 μl of Cold ATP mix in Assay Buffer, then 10 μl of a 1:10 dilution of α-Casein stock. Then add 2 μl of compound being tested (DMSO) at a 50× final concentration. Hot ATP mix (10 μl) is added, and the reaction is started with the addition of 10 μl of a 1:350 dilution of the ALK-5 protein (2 nM final) in Assay Buffer with 0.05% BSA (Bovine Serum Albumin). The reaction is mixed for 5 minutes at room temperature, and then continued for 145 minutes at room temperature. The reaction is then stopped with the addition of 100 μl of ice-cold 20% TCA (trichloroacetic acid). The assay is then incubated for at least 1 hour at 4° C., and then the contents of each well are filtered by suction through the filter. The wells are washed three times with 200 μl ice-cold 10% TCA. The plate bottom is blotted before and after removing plastic sub-base, and dried overnight at room temperature. Add 30 μl of scintillation fluid, and count 1 minute per well on a Wallac Tri-Lux scintillation counter.
Affinity data for this assay
 

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Last update November 1, 2007
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