Bio-Rad DC™ Protein Assay - Manual (Page 5)

Section 5
Instructions
5.1 Standard Assay Protocol
1. Preparation of working reagent
Add 20 µl of reagent S to each ml of reagent A that will be needed for the run.
(This working reagent A' is stable for one week even though a 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, thereby altering the volume of reagent that is added to the sample.)
If samples do not contain detergent, you may omit step #1 and simply use
reagent A as supplied.
2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to
about 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 of reagent A' or A (see note from step 1) into each test tube.
Vortex.
5. Add 4.0 ml reagent B into each test tube and vortex immediately.
6. After 15 minutes, absorbances can be read at 750 nm. The absorbances will
be stable at least 1 hour. (See Troubleshooting Guide for recommendation
on using a wavelength other than 750 nm.)
5.2 Microplate Assay Protocol
1. Preparation of working reagent
Add 20 µl of reagent S to each ml of reagent A that will be needed for the run.
(This working reagent A' is stable for 1 week even though a precipitate
will form after 1 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,
thereby altering the volume of reagent that is added to the sample.)
If samples do not contain detergent, you may omit step #1 and simply use
reagent A as supplied.
2. Prepare 3 - 5 dilutions of a protein standard containing from 0.2 mg/ml to about
1.5 mg/ml protein. A standard curve should be prepared each time the assay
is performed. For best results, the standard should be prepared in the same
buffer as the sample.
3. Pipet 5 µl of standards and samples into a clean, dry microtiter plate.
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