Prior to beginning, place sonicator arm in cold room to allow it to equilibrate to 4C. Get a cooler with LN2 and precool tubes for powder, weigh tools and large tweezers. Get a bucket of ice to cool your wash buffers. Samples are to be kept on ice at all times unless otherwise noted.
Sampples: Normal liver (N1) FLC (T1)
Extraction/Wash buffers: 20mM HEPES Na pH7.4, 500mM NaCl, 1% Triton X-100
Wash buffers should be kept on ice; extraction buffer with protease inhibitors kept at RT
Label and precool 2ml safe lock eppendorf tubes in LN2 Weigh out 100 mg of powder for each Sample
Allow powder to sit at RT for 1 minute before adding buffer Add 400ul of extraction buffer (RT, with 100X protease inhibitor added) to each sample, Vortex to resuspend. If powder is not resuspended after 10 seconds, return sample to ice for 30 seconds before additional vortexing to prevent temperature increase
Sonicate 2 x 10 Seconds, 4 Amp, expected energy output is 15J; Record output readings for each sample Spin @ ~21k rcf (top speed of benchtop eppendorf 5424R), at 4C for 10 minutes.
Wash beads while spinning Place empty 1.5 ml safe-lock tubes on magnet; add 0.5ml of appropriate wash buffer to tubes Add 7ul anti-PRKACA beads to each tube ( New and Old beads) Close tubes and remove tubes from magnet, vortex and quick spin to get beads from top of tube. Aspirate buffer with vacuum
2x 0.5ml additional washes (3 total), change tube on the second wash. After spin, save 20ul of clarified whole cell lysate (Input).
Use remainder of clarified whole cell extract to setup IPs (half to old beads and the rest added to new beads. IP incubation with end-over-end mixing in cold room for 30 min Wash the beads with 3x 0.5 mL of appropriate wash buffer.
Switch beads to new tubes during second wash step to ensure nothing carries over from IP to eluate. After the 3rd wash, give beads a final quick spin to remove last ul of wash buffer
Elute the beads with 19 ul of 1.1X LDS, 70 °C for 5’ with mixing, 1000rpm. Collect the elution on Magnet.(Elution) Add 1ul of 1M DTT to final concentration 50mM.
Heat at 70℃, 10min Spin to get rid of bubbles. Load all elutions and BSA to 15-well 4-12% Bis-Tris gel, save 2ul for WB run 45min using MOPS running buffer.
Note : The antibody used in the NEW PRKACA AB (0.2mg batch).