Analysis of Serum Proteins by LC-MS/MS - LC-MS/MS in Proteomics: Methods and Applications - page 288

single samples or pooled serum samples can be used. Ideally,
Eppendorf Lo-bind protein tubes (Eppendorf UK Limited,
Histon, UK) should be used throughout the whole protocol.
2. It is best to aliquot the 10X buffers (5 mL for Equilibration
and 2.5 mL for elution buffer) into 50 mL Falcon tubes
under sterile conditions. The elution buffer may be cloudy,
so warming it up for a couple of minutes as 37 ◦ C resolves
the precipitate. After diluting the 10X to 1X we recommend
a filter-sterilization step, using a 0.2

m syringe filter (Min-
isart Plus, Satorius) and 50 mL syringe. Further we recom-
mend performing all of the depletion steps under sterile con-
ditions to avoid contamination.
3. In our studies we collect the wash fraction separately and
concentrate it. We do not add this fraction to the depleted
fraction, as it dilutes it and does not contribute significantly
to the protein amount. If desired, the wash fraction may be
added to the depleted fraction.
4. In our laboratory, several methods were tested to concen-
trate the depleted protein fraction. We found that the use
of centrifugal filter concentration yielded minimal protein
loss and optimum protein quality. However, the alternative
approaches of precipitation, different types of centrifugal fil-
ter devices, and dialysis can be used.

μ

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