Biology Reference
In-Depth Information
Samples were applied adjacent to the acidic end of the IPG
strips by cup loading. IEF conditions were as follows: in a
1-h gradient up to 300 V, 1 h at 300 V, followed by a gradi-
ent of ~10 V/min up to 3,500 V, and fi nally 21 h and 26 min
at 3,500 V (20°C). Total running time: ~28 h and 30 min
(see Note 13).
5. Focused IPG strips can be stored at −20°C or directly applied
to the second dimension.
6. Prior to second-dimension separation, IPG strips were equili-
brated for 15 min in equilibration buffer.
7. Subsequently, IPG strips were re-equilibrated for 15 min in the
same buffer containing 4.5% (w/v) iodoacetamide instead of
DTT (see Note 6).
8. For protein separation by size in the second dimension, 12.5%
polyacrylamide gels containing 5% glycerol combined with a
Tris/glycine buffer system were used (thickness 1.0 mm).
Electrophoresis of up to 12 large-format gels (24 × 18 cm) was
conducted simultaneously at 3 W/gel and 20°C on an Ettan
Dalt 12 system. The run was stopped when the blue dye front
reached the end of the gels. Total run time: 16-19 h (see Note 7).
3.5. Imaging and
Image Analysis
1. Closed glass plates were rinsed with double-distilled water,
cleaned, and dried with lint-free paper. Gels were scanned
directly between the glass plates using a Typhoon 9400 (GE
Healthcare) laser scanner (see Note 14).
2. A resolution of 100
m was suffi cient for good quality results:
photomultiplier tube settings of 500 V (see Note 15).
3. The Cy2 pattern was scanned at a wavelength of 488 nm
(blue laser), Cy3 at 532 nm (green laser), and Cy5 at 633 nm
(red laser).
4. Image analysis and evaluation of protein spot patterns, abun-
dance, and statistics was accomplished automatically using
Proteomweaver™ software (Bio-Rad). The gel images derived
from identical sample types were grouped for comparison
(see Note 16).
μ
4. Notes
1. Sample quality is one important factor and significantly
contributes to success or failure of biomarker discovery projects.
In case of blood samples, special attention has to be paid to the
collection tubes used (identical and appropriate tube types for
the entire study), mix ratios (fi ll to the specifi ed volume), and
the stability of incubation conditions (use constant times,
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