The solutions met immediately upstream of the emulsification nozzle, ensuring that transcription did not begin before the beads were compartmentalized within droplets.
We delivered C12-resazurin to pre-formed emulsion droplets [31] to ensure that the reaction did not begin before the beads were compartmentalized.
Samples were further incubated with A/G Pluss Agarose beads (Santa Cruz Biotechnology) at 4°C for 25 minutes before the beads were removed by centrifugation at 5000g for 4 minutes and the samples were incubated with 1.5 µg anti-V5 (Invitrogen) at 4°C for 90 minutes.
The isolated complexes were washed, aliquoted, and recombinant protein was then incubated with the 7SK snRNP for 15 minutes before the beads were concentrated and analyzed by Western blot for LARP7 as a control for loading and for Cdk9 to detect release of P-TEFb.
Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 5 min. Western analysis was performed using standard protocols and the following antibodies: EGFR (1∶1000, Santa Cruz), ErbB2 (1∶1000, Abcam), ErbB3 (1∶1000, Santa Cruz), ErbB4 (1∶1000, Santa Cruz); flag (1∶1000 M2, Sigma), and actin (1∶8000, Abcam).
Place tubes on a rotator at room temperature for 10 min. Spin the beads solution down before the beads dry permanently onto the wall of the tube.
To compensate for differences among the probes caused by different probabilities of DNA disruption and hybridization efficiency, signals were normalized by dividing them by the signal intensities from DNA samples taken before the beads were added.
To minimize nonspecific binding of other proteins during the immunoprecipitation, all plasma samples were pretreated twice with 20 μl of Novex Dynabeads Protein G (Life Technologies, Carlsbad, CA, USA) per milliliter of plasma and incubated for 1 hour at RT before the beads were magnetically removed as described previously, with some modifications [ 20].
A 25 μl of glutathione Sepharose was added and samples were incubated for 3 h at 4°C with rolling before the beads were removed by centrifugation (10,000 g, 10 min, 4°C) and the supernatants were used directly for primary antibody immunoblot incubations.
