This protocol was used in our Nature Immunology paper.
Method Article
Immunoprecipitation of endogenous MEKK1 and kinase assay
https://doi.org/10.1038/nprot.2007.38
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This protocol was used in our Nature Immunology paper.
Hypotonic Buffer:
20 mM HEPES (Sigma) pH 7.6, 10 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA (Sigma), 1 mM EGTA (Sigma), 0.5 mM PMSF (Sigma), 10 µg/ml of leupeptin (Sigma), 10 µg/ml of aprotinin (Sigma), 5 µg/ml of pepstatin A (Sigma), 50 mM β-glycrophosphate (Sigma).
22 gage needles
5 M NaCl
Protein A beads
Triton X-100 (Sigma)
Anti-Myc or anti-p65 (both Santa Cruz)
Anti-MEKK1 (4)
Liquid nitrogen (eventually)
For kinase assay only:
Kinase Assay Buffer:
20 mM HEPES pH 7.8, 10 μM ATP, 10 mM MgCl2, and 10 mM β-glycrophosphate.
JNKK1(K131M)
[γ-32P]ATP
Preparation of lysates
1 Harvest approximately 1×107 cells by centrifugation at 2000 RPM for 5 min. Aspirate media and resuspend cell pellet with 1 ml of ice cold PBS and transfer to a 1 ml centrifuge tube. Microcentrifuge at 2000 RPM for 5 min at 4 ˚C.
2 Aspirate PBS, and then add Hypotonic Buffer (supplemented with 1% Triton X-100, to disrupt membrane and cytoskeleton bound MEKK1 fractions).
3 Cell lysates are then homogenized by passing through 22 gage needles, and tubes are put on ice for 15 min to complete the lysis. Crude extracts are then centrifuged at 2500 RPM for 5 min. Supernatants are then transferred to fresh centrifuge tubes, and cold 5 M NaCl is added to each sample to make a salt concentration of between 0.7 – 1.0 M, in order to disrupt protein-protein interactions.
4 Spin the crude extracts by ultracentrifugation at 55000 RPM to properly pellet residual insoluble proteins from the extract. Transfer supernatants into fresh centrifuge tubes.
Immunoprecipitation
5 Rinse Protein A beads in Hypotonic Buffer and place on ice till ready for use.
6 Take a volume of cell lysates (prepared as described above), and dilute with Hypotonic Buffer to 250 – 500 mM salt to enable protein-protein interactions.
7 Add 2 µg of preclearing antibody to the diluted lysate (e.g. anti-Myc or anti-p65), vortex, add 50 µl of Protein A beads, and rock at 4 ˚C for 45 min.
8 Touchspin samples, and transfer supernatant to a fresh tube.
9 Next, add 2 µg of polyclonal anti-MEKK1 (4) to the lysates, and rock for 1 h. After this period add 50 µl of Protein A beads and rock tubes at 4 ˚C for 1 h.
10 Touchspin beads, wash beads with hypotonic buffer (supplemented with NaCl to a concentration of 300 mM), vortex, and rock for 10 min. In total, 3 – 5 washes of the beads are performed.
11 Finally, wash once with Hypotonic Buffer, and resuspend in Kinase Assay Buffer. Purified MEKK1 may be stored by snap freezing in liquid nitrogen and long-term storage at -80 ˚C.
Kinase assay
Following preparation of MEKK1 immunoprecipitates (as above), incubate with 7 µg of JNKK1(K131M) along with 5 µCi of [γ-32P]ATP in Kinase Assay Buffer for 30 min at 30 ˚C.
For immunoprecipitation, 4 h
For kinase assay, 30 min
Supported by the National Institutes of Health (ES04151 and ES06376 to M.K.), the American Cancer Society (M.K.) and the Leukemia and Lymphoma Society of America (T.E.; SCOR grant 7332-06).
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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