Tie2, active

It is important to shake the reaction mixture during incubations so that the substrate and the enzyme get good interaction. If you do not have access to a shaking incubator, use a water bath for the incubations, removing the tubes regularly to vortex.

The best buffer is Buffer A. It is composed of 50mM Tris/HCl, pH 7.5, 1mM EGTA, 1mM EDTA, 1mM Sodium Orthovanadate (activated), 10mM Na B-glycerolphosphate, 50mM NaF, 5mM Na pyrophosphate, 1% w/v Triton X-100, 0.1% w/v 2-mercaptoethanol, 1ug/ml each of aprotinin, leupeptin and pepstatin, 1mM PMSF and 1mM microcystin (this is optional). QS with water. You may store Buffer A in aliquots at -20°C for 6 months.

The Molar extinction coefficient is 15400.

In order to maintain activity of enzymes, we recommend a slow thaw and aliquot storage. To do this, remove the full vial from the freezer and place under cold running tap water. Once thawed, place the vial on ice, gently mix, than make aliquots. Using liquid nitrogen or an ethanol/dry ice bath, flash freeze the aliquots. Store aliquots at the recommended temperature.

The purpose of washing the p81 squares in acetone is mostly to help them dry faster, but it also can help to eliminate low specificity binding.

The role of Brij-35 is to function as a mild detergent, which prevents aggregation in active kinases.