Component	Amount per reaction			Final
	Reaction A	Reaction B	Reaction C
In vitro ubiquitinated substrate (from Step 1A)	4 µL	—	—	*
Immunoprecipitated substrate (from Step 1B)	—	10 µL	—	*
Heterotypic substrate (from Step 1A)	—	—	2-4 µg	2-4 µg
2x or 5x DUB (from Step 2)	5 µL of 2x	5 µL of 5x	20 µL of 2x	1x
10x DUB reaction buffer	1 µL	2.5 µL	4 µL	1x
MilliQ water	—	7.5 µL	To 40 µL
FINAL	10 µL	25 µL	40 µL

^*

Note: Actual concentration of these substrates is not known. The used amount is what is needed for clear visualization.