Protein Protocols Protein Protocols

PAGE Using Ampholyte pH Gradients 165
40 mL 10% (w/v) SDS stock solution
8 mL 0.05% (w/v) bromophenol blue stock solution
72 mL dH 2O
This solution can be stored at room temperature for 2 to 3 mo.
2.3. SDS-PAGE
1. The Protean II chamber (Bio-Rad) is employed by us for SDS-PAGE. The gels (160 × 200
×1.5 mm) are cast in the Protean II casting chamber (Bio-Rad). The gradient former is
model 395 (Bio-Rad).
2. Running buffer: 50 mM Tris base, 384 mM glycine, 0.1% (w/v) SDS in dH2O. For 1 L: 6 g
of Tris base, 28.8 g of glycine and 1 g of SDS. Do not adjust pH. Fresh solution is made up
for the upper tank. The lower tank running buffer can be retained for more than 6 mo with
the addition of 0.02% (w/v) sodium azide.
3. Sodium thiosulfate stock solution: 5% (w/v) sodium thiosulfate anhydrous in dH2O. This
solution should be stored at 4°C. (See Note 1).
4. Silver staining: Proteins in the 2-D gel are stained with silver. We used the ammoniacal
silver nitrate method described by Oakley et al. (5) and modified by Hochstrasser et al. (6)
and Rabilloud (7). (See also Chapter 33.)
3. Method
3.1. Preparation of Protein Samples
Pellets of cells or tissue should be resuspended in 100 µL of 10 mM Tris-HCl, pH 7.4,
and sonicated on ice for 30 s. Add one volume of lysis solution B and mix. The mixed
solution should not be warmed up above room temperature. Samples can be loaded
directly onto the gels or stored at –80°C until needed. (See Note 2 for plasma sample
preparation and Note 3 for optimal sample loading.)
3.2. Isoelectric Focusing (IEF)
1. Draw a line at 16 cm on clean and dry glass capillary tubes (capillary should be cleaned
with sulfochromic acid to eliminate all deposits). The remaining 0.5 cm of the capillary
tubes is used to load the sample. Place each tube in a small glass test tube (tube of 5 mL)
which will be filled with the isoelectric focusing gel solution. Connect the tops of each
glass capillary tube to flexible plastic tubes joined together with a 1-mL plastic syringe.
2. At least 12 capillary gels can be cast with 11.5 mL of isoelectric focusing gel solution
(800 µL of solution is needed per capillary).
a. Prepare 1 mL of CHAPS 30% (w/v) and Nonidet P-40 (NP-40) 10% (w/v) (0.3 g of
CHAPS and 0.1 g of NP-40 ) and degas for 5 min.
b. Separately, prepare a second solution: 10 g of urea (7 mL of water is added to dissolve
the urea at warm temperature, around 35°C), 2.5 mL of acrylamide stock solution,
0.6 mL of ampholytes, pH 4–8; 0.4 mL of ampholytes, pH 3.5–10; and 20 µL of
N,N,N'N'-tetramethylethylenediamine (TEMED).
c. Mix the first solution (CHAPS, NP-40) with the second one. Degas the mixture and
add 40 µL of APS 10% (w/v) stock solution.
Pipette 1 mL of this isoelectric focusing gel solution into the glass test tubes (along the
side walls in order to prevent the formation of air bubbles in the solution). Fill up the
capillary tubes by slowly pulling the syringe (up to the height of 16 cm).
d. After 2 h of polymerization at room temperature, pull the capillary tubes out of the
glass test tubes. Clean and gently rub the bottom of each capillary gel with parafilm.