Bio-Rad RC DC™ Protein Assay - Manual (Page 3)

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Section 4 Assay Instructions
Standard Assay Protocol (5 ml)
1 Add 20 µl of DC Reagent S to each 1 ml of DC Reagent A that will be needed
for the run. This solution is referred to as Reagent A´. Each standard or sample
assayed will require 510 µl of Reagent A´.
(Reagent A´ is stable for one week even though precipitate will form after one
day. If precipitate forms, warm the solution and vortex. Do not pipet the
undissolved precipitate as this will likely plug the tip of the pipet and alter the
volume of Reagent A´ added to the sample.)
2 Prepare 3-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml
protein. A standard curve should be prepared each time the assay is
performed.
(For best results, the standards should always be prepared in the same buffer
as the sample.)
3 Pipet 100 µl of standards and samples into clean, dry test tubes.
4 Add 500 µl RC Reagent I into each tube, vortex. Incubate the tubes for 1
minute at room temperature.
5 Add 500 µl RC Reagent II into each tube, vortex. Centrifuge the tubes at
15,000xg for 3-5 minutes.
6 Discard the supernatant by inverting the tubes on clean, absorbent tissue
paper. Allow the liquid to drain completely from the tubes.
7 Add 510 µl Reagent A´ to each tube, vortex. Incubate tubes at room
temperature for 5 minutes, or until precipitate is completely dissolved. Vortex
before proceeding to the next step.
8 Add 4 ml of DC Reagent B to each tube and vortex immediately. Incubate at
room temperature for 15 minutes.
9 After the 15 minutes incubation, absorbances can be read at 750 nm. The
absorbances will be stable for at least 1 hour.
Microfuge Tube Assay Protocol (1.5 ml)
1 Add 5 µl of DC Reagent S to each 250 µl of DC Reagent A that will be needed
for the run. This solution is referred to as Reagent A´. Each standard or
sample assayed will require 127 µl of Reagent A´.
(Reagent A´ is stable for one week even though precipitate will form after one
day. If precipitate forms, warm the solution and vortex. Do not pipet the
undissolved precipitate as this will likely plug the tip of the pipet and alter the
volume of Reagent A´ added to the sample.)
2 Prepare 3-5 dilutions of a protein standard from 0.2 mg/ml to 1.5 mg/ml
protein. A standard curve should be prepared each time the assay is
preformed.
(For best results, the standards should always be prepared in the same buffer
as the sample.)
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