Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 43)

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2-D Electrophoresis Guide Methods Chapter 8: Sample Preparation
Sample Quantitation (RC DC Protein Assay)
The RC DC protein assay is based on a modification of the Lowry protocol (Lowry et al. 1951) and is both
reducing agent compatible (RC) and detergent compatible (DC). Protein quantitation can be performed in
complex mixtures including 2-D sample solution. It involves addition of detection reagents to a protein solution
and subsequent measurement of absorbance at 750 nm with a spectrophotometer. Comparison to a standard
curve provides a relative measurement of protein concentration.
Add 127 µl of Reagent A ´ to each tube
and vortex. Incubate tubes at room
temperature for 5 min, or until the
precipitate is dissolved. Vortex.
Add 1 ml of DC Reagent B to each tube
and vortex immediately. Incubate at
room temperature for at least 15 min,
but no longer than 1 hr.
Read absorbance of each sample at
750 nm. The absorbances are stable
for at least 1 hr.
Plot absorbance measurements as
a function of concentration for the
standards.
Interpolate the concentration of the
protein samples from the plot and
sample absorbance measurements.
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Add 5 μl of DC Reagent S to each
250 μl of DC Reagent A needed.
This solution is referred to as
Reagent A
´ . Each standard or sample
assayed requires 127 μl Reagent A ´ .
Prepare 3–5 dilutions of a protein
standard (0.2–1.5 mg/ml protein).
Use distilled or deionized water as
the diluent.
Pipet 25 µl of protein standard
or sample into clean 1.5 ml
microcentrifuge tubes. Add 125 µl
of RC Reagent I into each tube and
vortex. Incubate the tubes for 1 min
at room temperature.
Add 125 µl of RC Reagent II into
each tube and vortex. Centrifuge
the tubes at 15,000 x g for 5 min.
Position the tubes with the cap
hinge facing outward.
Remove the tubes as soon as
centrifugation is complete. A small
pellet should be visible on the
hinge side of the tube. Decant the
supernatant. Reposition the tubes as
before. Briefly centrifuge again to bring
any remaining liquid to the bottom of
the tube. Use a micropipet to remove
the remaining liquid.
Microfuge Tube Assay Protocol (1.5 ml)
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