Biology Reference
In-Depth Information
(c) Another approach is to add to a TUC desorption solution
some amounts of cysteic acid [
40
].
(d) When followed by SDS-PAGE analysis, the elution could
be performed by boiling the beads with 200
L of Laemmli
buffer comprising sodium dodecyl sulfate and reducing
agents such as 2-mercaptoethanol or dithiothreitol.
μ
3.4 Fractionated
Elution
Desorption of proteins is obtained by successive washings with
solutions of increased stringency [
23
].
1. As fi rst eluting agent use 200
L of neutral 1 M sodium chlo-
ride solution to dissociate all proteins captured by a dominant
ion-exchange effect.
2. Then use as second eluting agent 200
μ
L of 60 % ethylene
glycol in water to dissociate proteins predominantly captured
by hydrophobic association.
3. Then incubate the CPLL with a third desorption solution
composed of 0.4 M glycine-HCl buffer (pH 2.5).
4. Finally all residual proteins still present on the beads are
stripped out by a mixture composed of 6 % acetonitrile, 12 %
isopropanol, 10 % of 17 M ammonia, and 72 % distilled water
(vol/vol).
In between desorption steps the CPLL beads could be rap-
idly washed with 200
μ
L of distilled water with a small risk of
desorbing some proteins. If this washing operation is not per-
formed then some overlap of proteins persists between fractions.
5. Individual fractions are then desalted, cleaned, or used directly
for further analysis (
see
Notes 4
and
5
).
μ
3.5 Direct On-Bead
Protein Digestion
When the analysis of captured proteins is performed by the so-called
shotgun approach, the most direct way to proceed is to make a
digestion of the captured proteins directly on the beads [
36
]. The
operation requires some excess of trypsin since part of it will be
captured by the CPLL beads. Basically the process is as follows:
1. After protein capture on the peptide library beads (whatever
the method or the physicochemical conditions), the beads are
rapidly washed twice with 200
L of 100 mM ammonium
bicarbonate containing 0.1 % Rapigest (this is not mandatory,
but it facilitates the proteolysis process). This is obtained by
adding 1 mL of 100 mM ammonium bicarbonate to the 1 mg
Rapigest vial lyophilizate and shake gently for few minutes.
The bead suspension is then vortexed for few minutes.
2. Then 300
μ
L of 10 mM DTT are added and the bead suspen-
sion heated at 65 °C for 1 h under gentle stirring or occasional
shaking.
3. 300 other
μ
L of 55 mM iodoacetamide are added, mixed and
stored in the dark for 60 min.
μ
