Biomedical Engineering Reference
In-Depth Information
containing the same master spot numbers as provided by
BVA quantitative analysis.
(3) The relative position of differentially expressed protein
spots that require identification are defined according to
the reference markers initially applied on the smaller gel
plate. Pick list coordinate file is exported to the Ettan auto-
mated spot picker.
(4) A gel containing spots of interest is clamped down into
position in the picker and submerged into 1-2 mm deep
layer of Milli-Q 18
water. The imported pick list is
opened and the spot picking head of the instrument aligned
to the reference markers previously defined by DeCyder
according to the manufacturer's instructions.
(5) Spots of interest are excised using a 2 mm picking head
and are placed in a designated 96-well plate in 200
μ
lof
Milli-Q 18
water. The water from each well is removed
prior to storage at
20
◦
C or subsequent digestion and mass
spectrometric analysis is performed.
−
3.5. In-GelTryptic
Digest
(1) Gel spots are dehydrated and destained by washing three
times in 30
l of 50% acetonitrile (ACN) and then dried
in SpeedVac for 10 min.
(2) Destained spots are removed from the 96-well plate and
transferred to labelled lo-bind Eppendorf tubes (most
96-well plates are not suited to tryptic digests as they bind
proteins and peptides).
(3) Di-sulphide bonds in proteins are reduced by the addition
of 10 mM DTT in 10 mM ammonium bicarbonate (pH
8) followed by incubation for 45 min at 50
◦
C.
(4) The DTT is removed and 50 mM solution of IAM in
10 mM ammonium bicarbonate (pH 8) is added followed
by incubation for an hour in the dark to alkylate cysteine
moieties in proteins.
(5) The IAM solution is removed and the gel pieces washed
twice in 50% ACN and dried in SpeedVac for 10 min.
(6) Sufficient trypsin solution to a total of 50 ng (conc. of
5ng/
μ
spot. Tubes are then incubated at room temperature for
10 min prior to overlaying each spot with 10-20
μ
μ
lof
10 mM ammonium bicarbonate.
(7) The samples are incubated at 37
◦
C overnight (not exceed-
ing 16 h).
(8) The samples are centrifuged at 10,000
×
g
for 2 min prior
to the addition of 5
l of 50% of ACN/5% trifluoro-acetic
acid. Samples are gently agitated for 5 min.
μ
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