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|English to Polish translations [PRO]|
Science - Biology (-tech,-chem,micro-)
|English term or phrase: bath control|
input - 3 x 4 PL stopped assay on Immobilon membrane, not washed
background (3 wells) - assay with H2O instead of enzyme
positive control (4 wells) - 3% DMSO instead of compound
bath control (1 well) - no reaction mix
Measure of enzyme activity: Tyrosine protein kinase assays with either purified GST-wtRET or GST-RETMen2B protein are carried out in a final volume of 30 PL containing 15 ng of either GST-wtRET or GST-RET-Men2B protein, 20 mM tris-HCl, pH 7.5, 1 mM MnCl2, 10 mM MgCl2, 1 mM DTT, 3 Pg/mL poly(Glu,Tyr) 4:1, 1% dimethyl sulfoxide (DMSO), 2.0 PM ATP (g-[33P]-ATP 0.1 PCi). The activity is assayed in the presence or absence of inhibitors, by measuring the incorporation of 33P from (g33P] ATP into poly(Glu,Tyr) 4:1. The assay is carried out in 96-well plates at ambient temperature for 15 minutes under conditions described below and terminated by the addition of 20 PL of 125 mM EDTA. Subsequently, 40 PL of the reaction mixture are transferred onto lmmobilon-PVDF membrane (Millipore) previously soaked for 5 minutes with methanol, rinsed with water, then soaked for 5 minutes with 0.5% H3PO4 and mounted on vacuum manifold with disconnected vacuum source. After spotting all samples, vacuum is connected and each wellrinsed with 200 PL 0.5% H3PO4. Membranes were removed and washed 4 x on a shaker with 1.0% H3PO4, once with ethanol. Membranes are counted after drying at ambient temperature, mounting in Packard TopCount 96-well frame, and addition of10 PL/well of Microscint TM (Packard). IC50 values are calculated by linear regression analysis of the percentage inhibition of each compound in duplicate, at 4 concentrations (usually 0.01, 0.1, 1 and 10 PM). One unit of protein kinase activity is defined as 1 nmole of 33P ATP transferred from [g33P] ATP to the substrate protein/minute/mg of protein at 37°C.
2 hrs confidence: 11 days confidence:
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