Protein Protocols Protein Protocols

DIGE 187
2.2. IEF
Fig 1. The two cyanine dyes used in DIGE.
1. The IEF equipment is a Multiphor II from Amersham-Pharmacia (Peapack, NJ), with the
DryStrip kit installed. The Teflon membranes are from the YSI (Yellow Springs, OH)
model 5793 standard membrane kit for oxygen electrodes. Light paraffin oil was from
obtained from Amersham-Pharmacia.
2. Rehydration buffer:
2% CHAPS 0.4 g
8 M Urea 9.6 g (same composition as lysis buffer; see Note 1)
6 M Urea 7.2 g
2 M Thiourea 3.0 g
2 mM Acetic acid 2.7 µL of glacial acetic acid (~17 M)
10 mM DTT 200 µL of 1 M stock or 0.031 g solid
1% Pharmalyte 500 µL of 40% Pharmalyte stock solution*
Make up to 20 mL with H 2O; store at 4°C (see Notes 5 and 6).
*Use the appropriate IPG solution which corresponds to the pH range of the IEF gels. See
Subheading 3.2.
3. Equilibration buffer I:
1× Stacking gel buffer 25 mL of 4× solution
1% SDS 20 mL of 10% SDS solution
8.7% Glycerol 10 mL of 87% solution
5 mM DTT 500 µL of 1 M solution or 0.076 g
Bring up to 100 mL with H 2O see (Note 5).
4. Equilibration buffer II:
Same as above, except 2% iodoacetamide (add 2.0 g) replaces DTT, and a trace amount of
bromophenol blue is added.
2.3. SDS-PAGE
1. SDS is available from Fisher Scientific. The gradient maker, acrylamide and bis-acrylamide
of the highest purity are available from Bio-Rad (Hercules, CA). The gel equipment was a
Hoefer SE-660 18 × 24 cm apparatus from Amersham-Pharmacia. The 0.2-µm filters are
available from Nalgene (Rochester, NY).
2. 4× Resolving gel buffer: Dissolve 36.3 g Tris in 150 mL H2O. Adjust to pH 8.6 with 6 N HCl
then make up to 200 mL with H2O. Filter sterilize through a 0.2-µm filter and store at 4°C.
3. 30% Monomer solution: 60.0 g of acrylamide, 1.6 g bis-acrylamide made up to 200 mL
with H2O. Filter sterilize and store as described.