PRKACA- Test IP/Western
Date: 13/03/2021 Progenitor protocol: https://www.jove.com/video/54518/protein-complex-affinity-capture-from-cryomilled-mammalian-cells
Sample: FLC-PDX
Scale, 100mg with 7ul of anti-PRKACA beads Extraction/wash buffers:
3) 20mM HEPES, pH7.4, 1% (v/v) Tritonx-100, 500mM NaCl
Extraction buffers supplemented with 100x protease inhibitors, Roche, Cat # 11873580001 Extraction:
3x 100mg of FLC tumor and 3x 100mg of matched normal tissue
Allow powder to sit at RT for 1 minute before adding buffer
Add 400ul of RT extraction buffer (with protease inhibitor) to each sample Sonicate @ 4AMP for 5x2 seconds; energy output ~15J Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5424R) Save the pellets for extraction and 10% of supernatant (“Input”) for Western During the spin prepare the beads: Place tubes on magnet; add 1ml of wash buffer to each tube Add 7ul of bead slurry to each tube; vortex beads very well before beginning and before each addition Wash 2x more 1ml per wash
Add the lysate (~440 ul) to the equilibrated beads IP@ 4°C for 30’ Spin and collect 10% of unbound fraction aka flowthrough for Western (“FT”) Wash 3x 1ml cold extraction buffer (transfer the beads to fresh tubes during 2nd wash) Spin beads very briefly after 3rd wash to remove last ul of wash buffer Elute with 28ul of 1.1xLDS @70°C for 5’ with agitation (e.g. on thermo mixer, 1000rpm), save 10% (2.8ul) of the elution for Western (”E”) Save all fractions at -20C for gels ALEX - STOP HERE All samples to be combined with 50 mM DTT (1x), heated @ 70°C for 10’ with then cooled and spun before loading.