Biology Reference
In-Depth Information
every diluted sample as little pH adjustment might become
necessary in some rare cases.
11. This protein assay used tolerates many chaotropes and deter-
gents including urea and CHAPS.
12. It is essential to reconstitute the CyDyes with anhydrous DMF
(see Note 5) to a 1 nM-stock solution. If not used completely,
aliquots (make sure that the vessels are sealed properly) can be
stored at −20°C for several months without losing perfor-
mance. In our hands, a working solution of 400 pM (prepare
fresh before starting labeling) is suffi cient for almost all cases.
Tip: in the literature, some alternative dyes for protein label-
ing are described which may help to reduce the costs signifi -
cantly (
12, 13
).
13. In our hands, we achieve best results for IEF with the classic
Multiphor system (GE Healthcare) and a maximum voltage of
3,500 V instead of using state-of-the-art systems (e.g.,
IPGphor, protean IEF cell) with voltages of up to 10,000 V.
We use an extended starting time at low voltages to reduce
impurities that interfere with higher voltage focusing. As a
consequence, our standard protocols for most pH ranges take
21-30 h, and usually, we prefer to run IEF overnight. Evaluation
of optimized running conditions should be done for each
different sample types (e.g., cell lysates, organelles, plasma).
14. If scanning between the glass plate assemblies is preferred, we
recommend the use of a laser scanning system with confocal
optics to minimize artifi cial background signals arising from
the glass plate surface.
15. 500 V should be taken as initial value which has to be adjusted
specifi cally for each wavelength to achieve reproducible results.
Make sure that intensities in all three channels do not reach
saturation.
16. Usually a DIGE approach is based on the comparison of at
least two different samples. To minimize the impact of tech-
nical variation on experimental results, “dye swap” is recom-
mended. Therefore, each sample has to be labeled with both
dyes, i.e., Cy3 and Cy5. The differently labeled samples then
have to be separated (together with a second sample and the
internal standard) on different gels. In order to obtain statisti-
cally meaningful results, it is essential to analyze at least three
different biological (not technical) replicates of each sample.
Otherwise only experimental variability may be monitored and
not the biological changes. Remember that the internal standard
is composed of equal amounts of all the samples used in the
experiment and has to be calculated from the number of samples
and total number of gels to be run within the experiment. Tip:
due to the immense biological variability, for profi ling of
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