Application of 2D DIGE in Animal Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 395

50. If your cells are adherent, do not “trypsinize” them but scrape
them off the culture plate/fl ask with a cell scraper.
51. Any supernatant will dilute your labeling buffer and may infl u-
ence the unfolding of the proteins and the salt composition in
your sample.
52. Instead of DIGE labeling buffer, cells may be disrupted by
detergent-containing or low-salt buffer.
53. Samples should be put in an ice bath, either during homogeni-
zation or between different (short) homogenization cycles.
54. Tissues may also be homogenized in other solutions, especially
for fractionation purposes (e.g., differential solubilization ( 18 )
and subcellular fractionation ( 19 )). In addition, DIGE label-
ing buffer does not work equally well for all types of organ
samples. For kidney, for instance, it is better to use Tris buffer
with Triton X-100.
55. Optimize your assay to achieve high sensitivity, so that you will
be able to use high sample dilutions. In any case, for the blank
and the calibration curve use a similarly diluted sample buffer.
This will reduce the interference of additives.

56. In-gel rehydration may harm sensitive proteins as they are exposed

for hours to room temperature (during the swelling period) and
to different pH regimes (in the IPG strip). In addition, not all of
them may enter the gel only by passive re-swelling.

References

1. Westermeier R, Naven T, Höpker H-R (2008)
Proteomics in Practice. A Guide to Successful
Experimental Design, 2nd edn. Wiley-VCH
Verlagsgesellschaft GmbH, Weinheim,
Germany.
2. Uenlue M, Morgan ME, Minden JS (1997)
Difference gel electrophoresis: a single gel
method for detecting changes in protein
extracts. Electrophoresis 18, 2071-2077.
3. Timms JF, Cramer R (2008) Difference gel
electrophoresis. Proteomics 8, 4886-4897.
4. Miller I, Friedlein A, Tsangaris G, Maris A,
Fountoulakis M, Gemeiner M (2004) The
serum proteome of Equus caballus. Proteomics
4, 3227-3234.
5. Miller I, Teinfalt M, Leschnik M, Wait R,
Gemeiner M (2004) Nonreducing two-dimen-
sional gel electrophoresis for the detection of
Bence Jones proteins in serum and urine.
Proteomics 4, 257-260.
6. Kozlov AV, Duvigneau JC, Miller I, Nürnberger
S, Gesslbauer B, Kungl A, Öhlinger W, Hartl

RT, Gille L, Staniek K, Gregor W, Haindl S,
Redl H (2009) Endotoxin causes functional
endoplasmic reticulum failure, possibly mediated
by mitochondria. Biochim Biophys Acta - Mol
Basis Dis 1792, 521-530.
7. Miller I, Crawford J, Gianazza E (2006)
Protein stains for proteomic applications:
Which, when, why? Proteomics 6, 5385-5408.
8. Radwan M, Miller I, Grunert T, Marchetti M,
Vogl C, O'Donoghue N, Dunn MJ, Kolbe T,
Allmaier G, Gemeiner M, Müller M, Strobl B
(2008) The impact of Tyrosine kinase 2 (Tyk2)
on the proteome of murine macrophages and
their response to lipopolysaccharide (LPS).
Proteomics 8, 3469-3485.
9. Bradford MM (1976) A rapid and sensitive
method for the quantitation of microgram quan-
tities of protein utilizing the principle of protein-
dye binding. Anal Biochem 72, 248-254.
10. Miller I, Eberini I, Gianazza E (2010) Other
than IPG-DALT: two-dimensional electropho-
resis variants. Proteomics 10, 586-610.