Biology Reference
In-Depth Information
temperature, and mixing/centrifugation conditions) in order
to provide the required reproducibility.
2.
In the experimental standard confi guration, ProteoMiner™
columns (high-capacity kit) are designed to process 1 mL of
sample. Although it might be challenging to gather these
amounts of serum or plasma, we believe that at least this
sample amount is needed to access low-abundance proteins for
the following reason. Due to the extraordinary dynamic range
(>10 orders of magnitude) of plasma/serum (
1
), it is almost
impossible to discover potential biomarkers with concentra-
tions lower than 10
3
pg/mL (e.g., tissue leakage proteins or
cytokines) from unfractionated serum samples. Even if rather
sensitive technologies are used at least 10 fmol of a single pro-
tein species is required. As a consequence, this protein amount
has to be present in the starting volume. Typical concentra-
tions of biomarkers discovered so far are in the range of 10
3
pg/
mL (e.g., prostate specifi c antigen). Assuming a molecular
weight of 25 kDa their detection requires at least 1 mL of
whole plasma/serum to supply a suffi cient concentration
(1 ng). Despite this calculation, the hexapeptide material can
easily be up- or downscaled to the available sample amount
and concentration. For small sample amounts, a low-capacity
kit for 200
L serum/plasma is available and in addition dry
bulk beads are offered for customized approaches. Recently, it
has been shown that proteins of very low abundance could be
identifi ed after loading large volumes (>1,000 mL) of CSF
sample (<0.5 mg/mL) to the hexapeptide library. The infor-
mation revealed from this proteome mapping approach could
be used to increase the quantity and quality of data revealed
from small-scale experiments using as little as <1 mg CSF
loaded to reduced volumes of hexapeptide beads (
9
).
μ
3.
Beside its use for protein enrichment, hexapeptide library
beads can also be used for a fi nal “polishing” step in the pro-
cess of protein purifi cation. Minor protein impurities are
trapped by the beads, whereas the high-abundant proteins can
be recaptured from the fl ow-through. In addition, this strat-
egy allows better characterization of trace amounts of protein
impurities by analyzing the protein fraction removed by the
bead particles (
10, 11
).
4.
To enhance effi ciency and reproducibility, batches of 2D
DIGE lysis and rehydration buffers should be produced and
stored in appropriate aliquots (1-2 mL) at −80°C. Required
amounts of DTT and IPG buffer have to be added just before
use. To prevent decomposing, one should avoid heating any
urea- or thiourea-containing buffer.
5.
Always use the highest quality for anhydrous DMF to reconsti-
tute the CyDyes. Ideally, include a 4-Å molecular sieve inside
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