Harvest approximately 1×10<sup>7</sup> cells by centrifugation at 2000 RPM for 5 min.
{"action": "harvest", "output": "", "device": ["centrifugation"], "force": ["2000 RPM"], "time": ["5 min"]}
Cell lysates are homogenized by passing through 22-gauge needles,
{"action": "homogenize", "output": "", "reagent": ["cell lysates"]}
tubes are put on ice for 15 min to complete the lysis.

Crude extracts are then centrifuged at 2500 RPM for 5 min.
{"action": "centrifuge", "output": "", "force": ["2500 RPM"], "time": ["5 min"]}
Supernatants are transferred to fresh centrifuge tubes,
{"action": "transfer", "output": "", "container": ["fresh centrifuge tubes"]}
cold 5 M NaCl is added to each sample to make a salt concentration of between 0.7 – 1.0 M to disrupt protein-protein interactions. #####%%%%% output "residual insoluble proteins" are used in line14
{"action": "add", "output": "residual insoluble proteins", "container": ["each sample"], "reagent": [""]}
Spin the crude extracts by ultracentrifugation at 55000 RPM to properly pellet residual insoluble proteins from the extract. #####%%%%% output "Hypotonic Buffer" are used in line18
{"action": "spin", "output": "Hypotonic Buffer", "device": ["ultracentrifugation"], "force": ["55000 RPM"], "reagent": ["residual insoluble proteins"], "time": [""]}
Transfer supernatants into fresh centrifuge tubes.
{"action": "transfer", "output": "", "reagent": ["supernatants"], "container": ["fresh centrifuge tubes"]}
Rinse Protein A beads in Hypotonic Buffer until ready for use. #####%%%%% output "use" are not used
{"action": "rinse", "output": "use", "reagent": ["Hypotonic Buffer"]}
Take a volume of cell lysates (prepared as described above), . #####%%%%% output "Hypotonic Buffer" are used in line22
{"action": "take", "output": "Hypotonic Buffer", "volume": ["cell lysates"]}
dilute with Hypotonic Buffer to 250 – 500 mM salt to enable protein-protein interactions #####%%%%% output "antibody" are used in line24
{"action": "dilute", "output": "antibody", "reagent": ["Hypotonic Buffer"]}
Add 2 µg of preclearing antibody to the diluted lysate (e.g., anti), vortex, add 50 µL of Protein A beads, . #####%%%%% output "polyclonal anti-MEKK1" are used in line26
{"action": "add", "output": "polyclonal anti-MEKK1", "reagent": ["antibody", "Protein A beads"]}
Add 2 µg of polyclonal anti-MEKK1 to the lysates, , add 50 µL of Protein A beads at 4 °C for 1 h. 10.
{"action": "add", "output": "", "reagent": ["polyclonal anti-MEKK1"], "container": ["the lysates"]}
Touchspin beads, wash beads with hypotonic buffer (supplemented with NaCl to a concentration of 300 mM), .
{"action": "rinse", "output": "", "volume": [""]}
In total, 3 – 5 washes of the beads are performed. #####%%%%% output "Hypotonic Buffer" are used in line32
{"action": "perform", "output": "Hypotonic Buffer", "device": [""]}
Finally, wash once with Hypotonic Buffer, .
{"action": "rinse", "output": "", "reagent": ["Hypotonic Buffer"]}
Purified MEKK1 may be stored by snap-freezing in liquid nitrogen . #####%%%%% output "M" are used in line36
{"action": "use", "output": "M", "reagent": ["", ""]}
Kinase assay Following preparation of MEKK1 immunoprecipitates (as above), incubate with 7 µg of JNKK1(K131M) along with 5 µCi of [γ-<sup>32</sup>P]ATP in Kinase Assay Buffer for 30 min at 30 °C.
{"action": "incubate", "output": "", "reagent": ["M"], "container": ["Kinase Assay Buffer"], "temperature": ["30 \u00b0C"], "concentration": [""]}