Buffer: L1 buffer
“Cell line: HEK 293 Rrp6-3xflag cell powder (Apostolis 2021)"
(https://macromolecule-child.rockefeller.edu/#HEK%20293%20Rrp6-3xflag%20culturing)
"Magnetic beads: poor quality anti-FLAG
Before beginning:
● place sonicator probe in cold room
● mix extraction buffer aliquots with protease inhibitors; place the remaining buffer on ice in preparation to wash beads. Extraction buffer is held at RT
● fill ice bucket with ice
● fill small cooler with liquid nitrogen; pre cool weighing tools (large tweezer, large spatula and small spatula)
● pre-cool Eppendorf benchtop centrifuge (5424R) to + 4oC for spin after sonication
Scale: 150 mg in 15 ul of anti-3xFLAG beads, 6 replicate
Immunoprecipitation
https://macromolecule-child.rockefeller.edu/#Immunoprecipitation%20with%20antibody%20conjugated%20Dynabeads%20from%20cell%20powder
S-trap:
https://macromolecule-child.rockefeller.edu/#MS%20sample%20preparation%20using%20S-Trap%20Micro%20Ultra-High%20Recovery%20protocol%20rev.%2005%2F27%2F21
after dried, re-suspension samples in 37.5 uL of 0.1%FA, 5% methonal. then take out 25 ul to be dried and re-suspenson in peptide diluent (908 device) for zipchip analysis.
for nanoLC-MSMS:
- run 6 replicate samples first (sytem bias)
- Pool 6 samples, each sample 6 ul and run 6 times (system bias)
for zipchip-MS:
- run 6 replicate samples first (sytem bias).
the result is here:
https://docs.google.com/document/d/1zqTN9o2H8tatMmbw6nH4aDpJ9PQEJQvQ/edit?usp=sharing&ouid=108359822129145506549&rtpof=true&sd=true
Raw data: