Human recombinant proteins were purchased commercially (see Section 2.1: Supplies and Reagents). For cell-free protein expression, clones from the Human ORFeome Collection were used as the source for the ORFs. Standard Gateway® recombination cloning was performed (Walhout LBH589 in vitro et al., 2000) (Invitrogen, Carlsbad, CA) to transfer the ORFs into a custom T7 driven cell-free protein expression

vector containing a C-terminal streptavidin binding affinity tag (SBP-Tag; Keefe et al., 2001) and an N-terminal VSV-G epitope tag. Expression reactions were performed using one of two transcription/translation coupled systems, the Rabbit Reticulocyte Lysate (TNT® T7 Quick for PCR DNA), or the PURExpress® E. coli based reconstituted system, all according to the manufacturer’s instructions. The expression plasmid

used was a derivative of the pETBlue-2 vector containing the aforementioned tags and sequences for Gateway® cloning. Commercial recombinant proteins, which are supplied in a variety of formats, concentrations and buffers, were passed over a PD SpinTrap G-25 Column to remove potentially incompatible buffer components (e.g. Tris buffer or residual glutathione used in purifying GST fusion proteins) and to unify the buffer conditions. In the case where proteins were supplied lyophilized, they were first dissolved to 1 μg/μL in water and then supplemented to 1 × PBS, pH 7.5, from a 5 × stock before column purification. The PD SpinTrap G-25 columns 3-Methyladenine ic50 were performed according to the manufacturer’s instructions (equilibration in 300 μL Morin Hydrate 1 × PBS; 70–130 μL loading of the manufacturer supplied or reconstituted protein). Following the desalting (buffer exchange), 1/4th volume of 5 × PBS was added to the eluate to ensure an adequate buffering capacity of the protein for the subsequent bead attachment steps. Note that for optimal results with some proteins (e.g. p53 and MAP4K4), the column buffer exchange step was omitted and the manufacturer supplied proteins were simply supplemented to 1 × PBS (either

from a 5 × stock or as detailed above for lyophilized proteins). Note that while a comprehensive analysis of all possible buffer conditions was not done, some proteins (e.g. antibodies) coupled more efficiently to the VeraCode™ beads using a MES buffering system (0.1 M MES, pH 4.7, 0.9% NaCl) instead of PBS. In this case, MES buffer replaced the PBS in the aforementioned steps. Recombinant protein concentration used for subsequent bead attachment was typically 0.1 μg/μL in the corresponding buffer (if this concentration was not possible based on how the protein was supplied by the manufacturer, concentration was kept as high as reasonably possible). Capture antibodies to be coupled to VeraCode™ beads were not desalted, but were simply supplemented to 1 × concentrated MES Buffer and used at 0.5 μg/μL for subsequent bead attachment.