Table 1. iSAT protein synthesis of reactions containing combinations of crowding and reducing agents.

Relative mRFP1 production	No crowding agent	Ficoll 70 (6% w/v)	Ficoll 400 (6% w/v)	PEG 6000 (2% w/v)	PEG 8000 (4% w/v)
No reducing agent	1.0	2.1	2.2	2.0	2.0
DTT (2mM)	2.5	3.6	4.0	3.2	3.3
DTBA (2mM)	2.6	3.9	4.3	3.5	3.4
TCEP (2mM)	2.5	3.8	4.0	3.2	2.7

Crowding and reducing agents were included in iSAT reactions in combinations of concentrations with the greatest improvement on iSAT protein synthesis (Figure 2). Reactions were incubated at 37°C for 18 h and synthesis of mRFP1 was measured. mRFP1 production is shown relative to reactions with no additional crowding or reducing agents. Values represent averages (n ≥ 3) with 1 s.d.