Bio-Rad Experion Protein Analysis Kits - Manual (Page 23)

Starter Kit Test 2 — Percentage Determination
2.1 Overview of Test 2
In this test, you examine the separation of the BGG standard under reducing conditions and
different total protein concentrations. Under reducing conditions, BGG separates into light-
and heavy-chain fragments. These fragments are used to highlight the percentage
determination function of Experion
™ software, which calculates each fragment as a percent
of the total protein species in a sample. This quantitation method is fast, reliable, accurate,
and easy to perform; it is particularly useful for routine quality control experiments to evaluate
the purity of known proteins.
In this test, note that the relative abundance of light- and heavy chain fragments does not
change, regardless of the total protein concentration analyzed.
2.2 Assay Procedure
1. Set up the electrophoresis station, equilibrate the reagents to room temperature, and
prepare the gel (G) and gel-stain solution (GS) as outlined in Sections 1.2.1–1.2.3. If
the G and GS have already been prepared, equilibrate them as detailed in Section
1.2.2.
2. This protocol uses 1,000 and 500 ng/µl dilutions of BGG. Label 2 microcentrifuge tubes
(1–2) and add 200 µl DEPC-treated water into each tube. Then add the following:
Tube 1 (1,000 ng/µl): 200 µl BGG stock solution
Tube 2 (500 ng/µl): 200 µl BGG dilution from tube 1
Note: If you have recently run Test 1 or 3 (within a month and using proper storage at 4°C),
you can use the 1,000 and 500 ng/µl dilutions prepared for those tests in this test.
3. Prepare the reducing sample buffer (“R”) as described in Section 1.2.5. For this test, do
not prepare nonreducing buffer (“NR”). Prepare fresh sample buffer each day that an
assay is run.
4. Prepare the samples S1 and S2 and the Pro260 ladder as described in Section 1.2.6,
except use reducing buffer (“R”) for preparing both the samples and the Pro260 ladder.
5. Prime and load the chip as described in Sections 1.2.7 and 1.2.8 and using the chip
layout shown in Figure 2.1.
S2 S1 S2 GS
S1 S2 S1 GS
S2 S1 S2 GS
S1 L GS G
Fig. 2.1. Chip layout for Test 2. S1–S2, sample numbers; GS, gel-stain solution; G, Pro260 gel; L,
Pro260 ladder.
6. Run the Pro260 analysis as described in Section 1.2.9. Enter the sample names and
information appropriate to the chip layout for Test 2 (Figure 2.1) .
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