Biology Reference
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acetonitrile, to remove the Coomassie stain. After drying, the spots
are reduced with DTT, and cysteines are then alkylated with IA.
After further washing and lyophilisation, the gel spots are digested
overnight with trypsin. Peptides are then extracted into a mixture
of acetonitrile and trifl uoroacetic acid and are dried again and
stored at −80°C until ready for nanoLC-MS/MS analysis.
Typical experimental conditions for the nanoLC-MS/MS analy-
sis are detailed ubiquitously in the literature. See Subheading
2.1
,
item 10 for details of the instrumentation used in this example
and Table
3
for typical results obtained from the comparison of
different mouse genotypes. Note that in one case in Table
3
more
than one gel was chosen for the analysis of the same protein spot.
4. Notes
1. It is important to use anhydrous DMF for dissolving the
fluorescent dyes. The DMF bottle should be sealed with a
septum (to minimise ingress of air). If it was opened more than
3 months ago, it should not be used.
2. Dilution of the mouse liver lysates in water will be needed to
bring the sample absorbance within the range of the calibration
standards. Generally a 10 times dilution will often suffi ce. This
step will also help to avoid any interference in the colorimetric
technique from high levels of urea present in the lysate.
3. Optimum pH values for labelling the sample are between 8
and 9. No pH adjustment was found to be necessary when
using the lysis buffer described in Subheading
2.2.1
for the
presented example. However, the pH of each set of samples
should be checked before proceeding.
4. The amount of protein of each sample that will be pooled is
equal to no. of gels*100 mg protein/no. of samples, which
in the presented example equals 50 mg per sample. If the
protein concentration has been normalised at 3 mg/mL, this
is then equivalent to a nominal value of 16.67 mL of each indi-
vidual sample to be labelled with Cy2. To avoid problems due
to volumetric losses, in the presented example, this nominal
volume of 16.67 mL is increased to 20 mL (60 mg). Volumetric
losses can easily occur due to factors such as sample frothing,
sample adhesion to pipette tips and tubes, evaporation and the
cumulative effect of very slight pipettor volumetric inaccura-
cies. However, it is essential that all of the gels contain the same
level of internal standard. Analogously, the volume (amount)
of individual samples taken for Cy3 or Cy5 dye labelling is
increased to 40 mL (120 mg).
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