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wash solution (high-purity, 0.1 N HCl), basic wash solution
(high-purity 0.1 N NaOH), and an SDS-MW size standard
(10–225 kDa, 16 mg/mL).
(c)
Protein IS
.—10 kDa, Part No. A26487 (Beckman
Coulter, Inc.).
(d)
Water
.—LC grade.
(e)
β-Mercaptoethanol
.—Part No. M7154 or M6250
(Sigma).
D. Preparation of System Buffer Trays and
Standard and Sample Solutions
(a)
To prepare the system buffer trays, follow the steps in
Figure
2016.15A
and load reagents into the system inlet (lower
left panel) and outlet (lower right panel). Use 6 × 6 buffer trays,
following the configuration illustrated in the panels.
(b)
Either weigh 135 ± 5 mg skim milk powder
(SMP; protein content around 37%) or 500 ± 20 mg infant
formula powder (protein content around 11%) into a 15 mL
centrifuge tube.
(c)
Dissolve the sample and dilute to a 5 mL volume with
deionized (DI) water. Mix each tube on a vortex mixer until
the samples are homogeneously dissolved. Each final solution
should contain about 10–15 mg/mL protein.
(d)
Prepare the sample running presolution by mixing 1%
SDS sample solution with 10 kDa IS peptide using an 84:1
ratio based on the total number of samples to be analyzed in the
sample set (90 μL/sample).
(e)
Pipet 10 μL of each sample solution into separate 2.0 mL
microcentrifuge vials.
(f)
Sequentially add 85 μL sample running presolution and
5 μL β-mercaptoethanol to each microcentrifuge vial. Mix well
before heating the vials in a water bath at 100 ± 5°C for 10 min.
Cool down to room temperature, then centrifuge for 1 min at
about 7000 rpm.
(g)
Mix on a vortex mixer before transferring each sample
into their corresponding injection vials.
E. Sample Analysis
(a)
Set up an optimized separation method for the batch
analysis of up to 24 samples at a time, including a buffer blank
(10 μL DI water), an MW size standard, and an SMP sample.
(b)
For each separation cycle (40 min), precondition the
capillary first with basic wash solution, followed by acidic wash
solution, DI water, and SDS gel buffer.
(c)
Introduce the samples electrokinetically by applying
voltage at –5 kV for 20 s.
(d)
Perform electrophoresis at constant voltage with
an applied field strength of –497 V/cm and the capillary
thermostatted to 25°C using recirculating liquid coolant.
(e)
The current generated should be approximately 27 μA.
(f)
Program the system to automatically replenish all
reagents through incremental increases in buffer array after
every eight cycles.
(g)
Test system suitability using the MWmarker. Acceptance
criteria for the system suitability are as follows: The migration
time of the IS should be 12.3 ± 0.5 min, and the migration
pattern and migration times of the seven MW markers (10, 20,
35, 50, 100, 150, and 225 kDa) should completely separate
within 30 min using this method.
See
Figure
2016.15B
.
(h)
Acceptance criteria for the separation cycle are as follows:
The migration time of the IS should be 12.3 ± 0.5 min, the degree
of baseline drop from the migration time of the IS to the peak
valley between the end of casein and the peak of immunoglobulin
heavy chain (Ig H) and bovine serum albumin (BSA) should be no
more than 25% of the height of the IS of the sample.
(i)
To integrate SMP and infant formula electrophoregrams
(e-grams), set the baseline at 0.4 min before the IS peak to
the valley between the end of the κ-casein peak and the Ig H
Figure 2016.15A. Preparation of system buffer trays.
230