Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 16)

Very high-concentration extracts, with protein levels
above the upper limit of the standard curve, require that
lesser volumes of them be assayed. In such cases,
increase the amount of 0.12 N HCl (nominal) used so that
the total volume is 100 µl.
• Add 3.5 ml of diluted dye reagent to each tube. Vortex gently.
•
Measure the absorbances at 595 nm (A
595
) after 5 minutes.
•
Use the standard curve generated in Step 4 to estimate the
concentration of the sample proteins from the A
595
measurements.
Representative Standard Curves. Standard curves
were generated as described above with serial dilutions of
bovine gamma globulin in the three extraction solutions
14
Sequential Extraction Standard Curve
Bovine gamma globulin, in micrograms
0 50 100 150 200 250 300
12
Solution 3
Reagent 1
Solution 2
1
0%
0.6
0.4
0.2
0
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