Component (stock concentration)	Amount per assembly (µL)		Final concentration
	Assembly A	Assembly B
Ubiquitin (5 mM)	2	100	50 µM / 2.5 mM
E1 (10 µM)	2	2	100 nM
UBE2L3 (450 µM)	1	1	2.3 µM
GST-NEDD4 (100 µM)	20	—	10 µM
NleL (60 µM)	—	30	9 µM
ATP (100 mM)	20	20	10 mM
Ubiquitin ligation buffer (20x)	10	10	1x
MilliQ water	145	37
Total	200	200