Biology Reference
In-Depth Information
2. All buffers for cation exchange chromatography are contained
in the iCAT Cation Exchange Buffer Pack.
3. Assemble the holder and set the cation exchange column
into it.
4. Wash the column by injecting 1 mL of cation exchange buf-
fer—clean at the rate of 1 drop/s.
5. Inject 2 mL of cation exchange buffer—load at the rate of 1
drop/s.
6. Inject the iTRAQ-labeled peptide mixture into the column at
the rate of 1 drop/s and collect the eluate.
7. Inject 1 mL of cation exchange buffer—load at the rate of 2
drops/s to wash the column, and collect the eluate.
8. Prepare 25, 50, 75, 100, 200, and 350 mM KCl solutions by
diluting cation exchange buffer—elute (350 mM KCl) with
load buffer (without KCl). Inject 0.5 mL of each solution into
the column at the rate of 1 drop/s in increasing order of con-
centration. Collect each eluate in a new 1.5 mL tube (a total of
six fractions).
9. Inject 1 mL of cation exchange buffer—clean (1 M KCl buf-
fer) at the rate of 1 drop/s. Collect the eluate in a new 1.5 mL
tube.
10. Evaporate the seven eluates (from
steps 6
and
7
) down to
50
μ
L using a centrifugal vacuum concentrator.
3.7 Desalting Using
a C-18 Column
1. Add 500
μ
L of 0.4 % FA to each of the concentrated peptide
mixtures.
2. Wash a C-18 column by injecting 2 mL of 75 % ACN/0.1 %
FA at the rate of 2 drops/s.
3. Inject 1.5 mL of 0.1 % FA at the rate of 2 drops/s.
4. Inject the iTRAQ-labeled peptide mixture into the column at
the rate of 1 drop/s. Collect the eluate and reinject. Collect
the second eluate in a new tube.
5. Inject 2 mL of 0.1 % FA at the rate of 1 drop/s to wash the
column.
6. Inject 0.6 mL of 75 % ACN/0.1 % FA at the rate of 1 drop/s
and collect the eluate in a new 1.5 mL tube.
7. Repeat
steps 2
-
6
for each fraction.
8. Evaporate all of the samples to dryness using a centrifugal vac-
uum concentrator.
9. Redissolve each of the iTRAQ-labeled peptide mixtures with a
small amount of loading buffer used in the LC-MS/MS sys-
tem. Transfer the samples into desiccated vials.
