Bio-Rad Experion Protein Analysis Kits - Manual (Page 27)

Starter Kit Test 3 — Absolute Quantitation Using a
Calibration Curve
3.1 Overview of Test 3
In this test, you create a calibration curve that you use to determine the concentration of a
standard protein. You evaluate the linearity of the curve and check sizing and reproducibility.
Experion
™ software reports the results from relative and absolute quantitation methods
side-by-side in the results table. The results of this test demonstrate how use of a calibration
curve can significantly improve the accuracy of quantitation.
3.2 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. Prepare a serial dilution of the bovine g-globulin (BGG, 2.0 mg/ml) supplied with the kit.
Label six 0.65 ml microcentrifuge tubes 1–6. Tubes 1–5 are calibrants for creating the
standard curve; tube 6 is the sample for analysis and quantitation (that is, your
“unknown”).
Add 200 µl DEPC-treated water into each of the five calibrant tubes. 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
Tube 3 (250 ng/µl): 200 µl BGG dilution from tube 2
Tube 4 (125 ng/µl): 200 µl BGG dilution from tube 3
Tube 5 (62 ng/µl): 200 µl BGG dilution from tube 4
For tube 6 (400 ng/µl), combine 25 µl DEPC-treated water with 100 µl BGG dilution from
tube 2.
3. Prepare both the reducing and nonreducing sample buffers (“R” and “NR”) as
described in Section 1.2.5. Prepare fresh sample buffer each day that an assay is run.
4. Prepare the samples and Pro260 ladder as described in Section 1.2.6. Use reducing
buffer (“R”) for preparing the Pro260 ladder L; use nonreducing sample buffer (“NR”) for
preparing samples S1–S6.
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 3.1.
S6 S6 S6 GS
S5 S4 S3 GS
S2 S1 S6 GS
S6 L GS G
Fig. 3.1. Chip layout for Test 3. S1–S6, 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 3 (Figure 3.1).
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