Pulse chase protocol
1) Prewarm medium (DMEM – met/ cys) and lucite labeling box (optional).
2) Wash cells 2x with DMEM – met/cys (or PBS), then add 5 ml of the same medium and
place in CO2 incubator for 30 min. (emptying the pools of endogenous met by starvation)
3) Add 2 ml fresh DMEM – met/ cys + 5 % dialyzed fetal calf serum + 35S -met
'Express' label (0.5 mCi ~ 50 µl label) for each p100 dish. Place in CO2 incubator for 1
hr (pulse). Remember to have activated charcoal in the box and in the incubator to
absorp the radioactive methionine.
4) Aspirate the hot labeling medium with a 5 ml pipette and dispose properly as liquid
radioactive waste. Wash 1x with warm PBS; aspirate. Add 5 ml of warm DMEM + 10
% FCS + 2 mM cold Methionine (prepare 100x stock: 200 mM Methionine) to all dishes
except the “0” time point. Start timer: You will be performing the chase for 1, 3 or 6 hrs.
5) Immediately collect the cells from the “0” time point. Wash one more time with 1x
PBS, then scrape cells carefully into 1 ml of ice cold PBS and transfer to an eppendorf
tube. Spin in a microcentrifuge at 4 ˚C for 45 sec. Aspirate PBS and store cell pellet at –
80 ˚C until all time points have been completed.
6) When all time points have been completed and cell pellets stored at –80˚C, extract the
pellets in parallel by addition of 0.8 ml TEB (NP40 extraction buffer) + 2 mM EDTA +
protease inhibitors to each tube. Extract 15 min on ice, then spin at 10,000g for 10 min. in
cold centrifuge.
7) Transfer supernatant (lysate) to a new eppendorf tube already containing the antibody
(e.g. 1 µl each of p53 antibodies DO-1 and PAb1801). Discard pellet.
Incubate a minimum of 2 hrs at 4 ˚C, then add 30 µl of protein A sepharose to capture the
immunocomplex. Incubate another 45 min at 4 ˚C with mixing. Wash the beads at least
three times with TEB, each time spinning 1 min at low speed in a microfuge. Collect the
washes since they’re readioactive. Elute by boiling the beads with SDS sample buffer for
5 min, then analyze the IP’s by SDS-PAGE and quantitate bands at each time point using
PhosphorImager.
IMPORTANT: Check: 1) Personal survey form
2) Radioactive vial sheet (dispose of vial if nothing remains)
3) Check sheet on incubator
4) Sheet for disposal of label (appr. 90 % liquid waste, the rest solid)