The plate was incubated as before.
A 1 100 dilution of sheep antimouse IgG-conjugated to magnetic DynaBeads (Life Technologies) in StartingBlock containing 0.05% tween (herein referred to as screening buffer) was incubated with the library for 1 h, and hits were isolated using magnetic pull-down.
To determine the degree of Raf contamination of Suc (substrate impurity), assay buffer was incubated with 20 μl dH20 and processed as described above.
As a control, an aliquot of broken cells in an equal volume of KSHM buffer was incubated in parallel.
Crude enzyme mixture in these pH buffers were incubated for 30 min at 50 °C.
After the buffer was changed to separation buffer, the mixture was incubated at 37°C for 10 min.
Thus, the purified fusion protein (1 μg), or the same volume of elution buffer as a control, was incubated with 1 mM of each substrate, namely Put, Spd or Spm, 50 μM Amplex Red reagent, and 0.1 U ml-1 horseradish peroxidase in 100 mM Tris HCl buffer (pH 8.0), at 37°C for 1 h.
As controls, fibronectin was incubated in buffer alone or in buffer with PAD that had been heat-inactivated at 95°C for 20 minutes.
In brief, fibronectin was incubated with buffer containing PAD or, as a control, buffer alone.
After addition of buffer B (buffer A with 2.5 mM desthiobiotin), the sample was incubated again under the same conditions as previously described.
