Dear all,

I am looking for the substrates of a specific protein. To answer my question, I overexpress a FLAG-tagged variant of my active or inactive protein in HEK cell, immunoprecipitate FLAG and perform kinase assays on the beads. For this, I add ATP in the tube, together with kinase buffer (10 mM MgCl2 and MnCl2 in the presence of 0.01% NP40). I can see that my protein is active and uses ATP very nicely.

To identify substrates, I add the beads that contain immunoprecipitated kinase to whole-cell lysates in the presence of 10mM MgCl2 and MnCl2. The lysates are made in 0.1% NP-40. However, as soon as I add these components together, my beads disintegrate and I see precipitation in my lysates. The kinase reaction becomes very cloudy and the beads aggregate.

Would you know what the reason is for this precipitation, that does not occur when I perform my kinase assays on the beads but only when I add the beads to whole cell lysates?