Method for Protein Subfractionation of Cardiovascular Tissues Before DIGE Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 290

m fi lter.
Add 0.5 g CHAPS, 0.2 g DTT, 0.5 mL of IPG buffer (match-
ing the pH of the IPG strips), trace of bromophenol blue and
top up to 100 mL with ddH 2 O. Mix well, then freeze 1-mL
aliquots at −80°C.
5. Equilibration buffer: 6 M urea, 2% (w/v) SDS, 30% (v/v) glyc-
erol, 50 mM Tris-HCl, bromophenol blue. To prepare 500 mL
of equilibration buffer, mix 150 mL of glycerol and 150 mL of
ddH 2 O and dissolve 180.17 g urea. Separately dissolve 10 g
SDS in 50 mL of ddH 2 O and fi lter through a 0.2-

of Amberlite, stir for 10 min, and fi lter through a 0.2- μ

m fi lter.
Add 16.75 mL of 1.5 M Tris-HCl buffer, pH 8.8, and a trace
of bromophenol blue to the glycerol/water/urea solution.
Finally, add the SDS solution and top up to 500 mL with
ddH 2 O. Store in 40-mL aliquots at −20°C.
6. Equilibration buffer with 1% (w/v) SDS.
7. Equilibration buffer with 4.8% (w/v) iodoacetamide.
8. IPG strips: Immobiline DryStrip gels (IPG, GE Healthcare).
9. Gel buffer: 1.5 M Tris-HCl, pH 8.8. To prepare 1 L of gel buf-
fer, dissolve 181.7 g Tris-base in 800 mL of water. Add 20 mL
concentrated HCl and stir for 3-4 h. Carefully adjust pH to 8.8
with diluted HCl. Fill up to 1 L with ddH 2 O and fi lter through
a 0.2-

μ

m fi lter. Store at 4°C and keep away from light.
10. Acrylogel 2.6 (40%) solution (37.4% acrylamide, 2.6% N , N ¢-
methylenebisacrylamide (VWR, Lutterworth, UK)). Store at
4°C in the dark.
11. Gel polymerization: To cast large-format polyacrylamide gels
(12% T (total acrylamide concentration), 2.6% C (degree of
cross-linking)), mix 187.5 mL of Acrylogel 2.6 (40%) solution
with 150 mL of 1.5 M Tris-HCl, pH 8.8, and 253.5 mL of
ddH 2 O. Finally, add 6 mL of 10% SDS, 252

μ

L of tetrameth-
ylethylenediamine (TEMED) and 2.4 mL of 10% ammonium
persulfate (APS). Stir for about 10 s, then pour the gel solution
into the gel caster. Leave enough space at the top for placing
the IPG strip.
12. Overlay solution: Two butanol and ddH 2 O are mixed at a 3:2
ratio. Wait until there are two phases. Use the upper phase as
overlay solution.
13. Gel storage solution: 50 mL of 1.5 M Tris-HCl, pH 8.8, and
2 mL of 10% SDS are mixed and topped up to a fi nal volume
of 200 mL with ddH 2 O.
14. SDS running buffer: Dilute 10× Tris-glycine running buffer
(Invitrogen, Carlsbad, CA; LC2675) with ddH 2 O to make 1×
and 2× running buffer.
15. Agarose sealing solution: 1% (w/v) low-melting point agarose
in SDS running buffer with trace of bromophenol blue.

μ