Biology Reference
In-Depth Information
5. Remove carefully upper phase (phenol) and precipitate pro-
teins with fi ve volumes of ice cold 0.1 M ammonium acetate in
100 % methanol at −20 °C for 16 h.
6. Centrifuge at 5,000 ×
g
and 4 °C for 15 min.
7. Wash the protein pellet thoroughly twice in 20 mL of 0.1 M
ammonium acetate in 100 % methanol followed with two
washes in ice-cold 80 % acetone and fi nal wash 70 % ethanol.
Mix for 10-15 min. Centrifuge and repeat washing procedure
(
see
Note 6
).
3.2 Isoelectric
Focusing
1. Resuspend protein pellet in rehydration solution.
2. Remove insoluble matter by centrifugation for 20 min at
14,000 ×
g
.
3. Determine protein concentration by Bradford assay [
8
].
4. Required amount of proteins add to 1.5 mL eppendorf tube,
add corresponding IPG buffer or Ampholyte and bring vol-
ume up to required volume with IEF extraction solution
(315
L of sample for 17 cm long IPG strips). Vortex and spin
for 5 min at max speed.
5. Transfer the protein solution into isoelectrophoretic focusing
tray and rehydrate for 1 h at RT.
6. Overlay strips with mineral oil (1 mL).
7. Place strip into isoelectric focusing and start isoelectric focus-
ing under these conditions.
(a) Active rehydration (10 h at 50 V).
(b) 100 V for 100 Vh.
(c) 500 V for 500 Vh.
(d) 8,000 V for 99 KVh.
(e) Hold at 50 V.
μ
3.3 SDS-PAGE
Electrophoresis
1. Remove IPG strips from focusing unit.
2. Incubate IPG strips in SDS equilibration buffer with 2 % (w/v)
DTT for 15 min.
3. Incubate IPG strips in SDS equilibration buffer with 2.5 %
(w/v) IAA for 15 min.
4. Rinse strips with running buffer and place onto 12 % acryl-
amide gels.
5. Overlay strips with 0.5 % agarose in running buffer with traces
of bromophenol blue.
6. Carry second dimension SDS-PAGE at 2W/gel for 16 h or
until dye migrated off the gel.
7. Following SDS-PAGE, wash gels in three times in water for
15 min and stain for at least 12 h with CBB.
