Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 15)

4 Prepare a standard curve covering the range of 2–280 µg.
• Dilute the BgG standard with extraction solution in a two-
fold (or other) dilution series from 14 to 0.1 mg/ml. TBP can
be omitted from the standard-curve dilutions.
The standard curves generated with gamma globulin in the
three extraction solutions are similar. For approximating
concentrations, it is acceptable to use a single standard
curve generated with the protein dissolved in only one of
the extraction solutions (or in water). For more accurate
determinations, prepare the dilution series in the individual
extraction solutions. The high-concentration standard can
be in water.
•
Place 80 µl of 0.12 N HCl (nominal) in each assay tube.
• Mix 20 µl of each dilution of BgG standard with the 80 µl
of 0.12 N HCl (nominal) in each separate assay tube. Mix
gently.
• Add 3.5 ml of diluted dye reagent to each tube. Vortex gently.
•
Measure the absorbances at 595 nm (A
595
) after 5 minutes.
•
Draw a curve of A
595
versus the amount of protein (µg) for
the dilutions of BgG.
The curve is nonlinear.
5 Assay the extracts in a manner analogous to the preparation
of the standard curve.
•
Place 80 µl of 0.12 N HCl (nominal) in each assay tube.
• Mix 20 µl of each extract with the 80 µl of 0.12 N HCl
(nominal) in each assay tube.
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