4. 50% (v/v) of Glycerol: mix 200 mL glycerol (!99.0%) and
200 mL H 2 O by stirring (see Note 17).
5. Equilibration buffer: 6 M urea, 2% SDS, 50 mM Tris–HCl,
pH 8.8, 30% (v/v) glycerol. Weigh 180 g of urea. Then, add
16.5 mL of 1.5 M Tris–HCl buffer, pH 8.8, 100 mL of the 10%
SDS solution, and 300 mL 50% glycerol solution. Once mixed,
transfer to a volumetric flask and rise volume up to 500 mL.
Make 10 mL aliquots and store them at À80
C.
6. Reducing solution: 50 mM DL-dithiothreitol (DTT) (!99.0%)
in equilibration buffer. Dissolve 38.56 mg DTT in 5 mL of
equilibration buffer (see Note 18).
7. Alkylating solution: 260 mM iodoacetamide (IAA) (!99.0%).
Dissolve 0.23 g IAA in 5 mL of equilibration buffer (see Note
19).
8. IPG dry strips.
9. Isoelectric focusing system (Ettan™ IPGphor™ or
equivalent).
10. Forceps.
11. Spatula.
2.4 Second
Dimension: SDS
Polyacrylamide Gel
1. Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8.
2. 40% Acrylamide.
3. 2% Bis acrylamide.
4. 10% (w/v) Sodium dodecyl sulfate (SDS) solution.
5. 10% (w/v) Ammonium persulfate (APS) solution: dissolved 1 g
APS in 10 mL H 2 O (see Note 20).
6. N,N,N,N
0 -Tetramethyl-ethylenediamine (TEMED) (see Note
21).
7. Anodic running buffer: 25 mM Tris-base, 192 mM glycine,
0.1% SDS, pH 8.3. (see Note 22). Prepare 10Â Tris/glycine
stock solution dissolving 30.3 g of Tris and 144 g of glycine in
1 L of double-distilled H 2 O. Prepare 1Â working solution by
mixing 500 mL 10Â Tris/glycine stock with 50 mL of 10%
SDS solution prepared as described in step 4. Add water up to a
volume of 5 L. Store at 4
C.
8. Cathodic running buffer: 25 mM Tris-Base, 192 mM glycine,
0.2% SDS, pH 8.3 (see Note 22). Prepare 1Â working solution
by mixing 200 mL 10Â Tris/glycine stock solution with
40 mL of 10% SDS solution. Add water up to a volume of
2 L. Store at 4
C (see Note 23).
9. Precision dual color protein standards (see Note 24).
10. Paper electrode wicks.
11. Filter papers.
232
Lucı ´a Me ´ ndez et al.