Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages

Table 1 Methods tested for alkaline IEF of T. cruzi proteins

Method	Sample application^a	Strip rehydratation	Equipment	Cathodic DTT pad	IEF program
					Voltage (V)	Time (h:min)	Volt-hours (Vh)
A	In-gel rehydration sample in 350 μL of SB1	12 h	IPGphor	no	50 μA/strip, all step-n-hold
					500	1:00	500
					1,000	1:00	1,000
					8,000	4:00	32,000
B	In-gel rehydration sample in 350 μL of SB1	12 h	Multiphor II	65 mM DTT	2 mA and 5 W all step-n-hold
					500	0:01	8
					3,500	7:30	26,250
C	Anodic cup-loading sample in 100 μL of SB1	12 h in SB1 without sample	Multiphor II	65 mM DTT	2 mA and 5 W all gradient
					300	3:00	450
					1,400	16:00	11,200
					3,500	5:00	8,750
D	Anodic cup-loading sample in 100 μL of SB2	12 h in SB2 without sample	Multiphor II	SB2	2 mA and 5 W all gradient
					300	3:00	450
					1,400	16:00	11,200
					3,500	5:00	8,750
E	Paper-bridge loading sample in 250 μL of SB1	10 h in SB1 without sample	Multiphor II	SB1	2 mA and 5 W all step-n-hold
					150	1:00	150
					300	2:00	600
					600	1:00	600
					3,500	10:30	36,750
F	Paper-bridge loading sample in 250 μL of SB3	10 h in SB1 without sample	Multiphor II	SB1	2 mA and 5 W all step-n-hold
					150	1:00	150
					300	2:00	600
					600	1:00	600
					3,500	10:30	36,750

^a 500 μg protein was applied in 6–11 linear IPG strips, 18 cm, at 20°C
SB1 = 2-DE sample buffer 1 (7 M urea, 2 M thiourea, 0.5% IPG buffer 6–11, 65 mM DTT, 2% Triton X-100, 10% isopropanol and bromophenol blue)
SB2 = 2-DE sample buffer 2 (7 M urea, 2 M thiourea, 0.5% IPG buffer 6–11, 1.2% DeStreak, 2% Triton X-100 and bromophenol blue)
SB3 = 2-DE sample buffer 3 (7 M urea, 2 M thiourea, 0.5% IPG buffer 6–11, 85 mM DTT, 2.5% Triton X-100, 10% isopropanol and bromophenol blue)

ISSN: 1477-5956