Biology Reference
In-Depth Information
1.
Normalise the sample protein levels to 3 mg/mL by the addi-
tion of lysis buffer as necessary. This step greatly simplifi es the
protein labelling procedure. Check that the pH of each sample
is between 8 and 9 (see Note 3).
2.
To prepare the internal standard for labelling, pool an equal
volume (amount) of 20 mL (60 mg) of each sample in a suitable
tube to give a suffi cient volume (amount) of internal standard for
all gels. Ensure the tube contents are well-mixed (see Note 4).
3.
To prepare the individual samples for labelling, transfer for
each individual sample twice the volume used in the previous
step (40 mL) into an Eppendorf tube for labelling with either
Cy3 or Cy5 dye (see Notes 4 and 5).
4.
Keep the samples on ice until you are ready to label them.
5.
Prepare the CyDyes by addition of an appropriate volume of
anhydrous DMF to each tube of the CyDye kit (see Note 6).
The dye concentration of the resultant solution should be
400 pmol/mL.
6.
Usually 100 mg of protein is labelled with 800 pmol of dye. In
the example presented, the amount of protein actually taken
was 120 mg, and so 2.4 mL of the appropriate CyDye solution
is added to 40 mL of the sample.
7.
As the total volume of the pooled internal standard is
20 × 20 mL = 400 mL (1,200 mg), this is then labelled by the
addition of 24 mL of Cy2 dye.
8. The tubes containing the labelled samples and internal standards
are vortexed briefl y, spun and kept on ice in the dark for 30 min.
9. To stop the labelling reaction at least an equal volume of 10 mM
lysine solution compared to the CyDye solution volume of
steps 6 and 7 should be added to each tube. However, for
convenience a higher volume, i.e. 5.6 and 56 mL respectively,
should be chosen to increase the total volume by 20% to 48
and 480 mL, respectively. All sample solutions now provide a
suffi cient number of 40 mL aliquots, each containing 100 mg of
sample, for ten gels to be run with two individual samples and
one internal standard.
10. The samples are mixed, spun and kept on ice in the dark for a
further 10 min and can then be either frozen and stored at this
stage, or further prepared for the rehydration of the IPG strips
(see Subheading
3.3
).
In this procedure, the labelled samples are fi rstly mixed in accor-
dance with the gel plan (see Table
1
) and then reduced with DTT.
Modifi ed DeStreak solution is added to make a total volume that
is compatible with the IPG strip size and method of rehydration.
3.3. Rehydration
of IPG Strips
Search WWH ::
Custom Search